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   Text book of Biochemisty by u satayanarayana Text book of Biochemisty by u satayanarayana Presentation Transcript

  • Dr, lJ, Satyanarayana M.Sc,.Ph.D.,F.l.C.,F.A.C.B. Professor of Biochemistry Siddhartha Medical Colle g e (NTR University of Health Sciences) Vijayawada, 4.P., India Dr, lJ, Chakrapani M.B.B,S.,M.S. BCDCDT(SAn|D ALLTED lPf Ltd. No.1-E(1) "SHUBHAMPLAzA" (lst Floor) 83/I, BBLrRcrnrn MarN Roeo, Korrere 700010 (Ixora) k:i : (+9| -33)6535-3844,2241-8573 oFax : (033)2358-2127 e-mail : books@cal.vsnl.net.in
  • Eiochemistrg First Published : March 1999 Reprinted: 1999 RevisedReprint: August 2000 Reprinted: 2OQO,2001, 2QO2 Second Revised Edition : June 2002 Reprinted: 2003 RevisedReprint: 2004 RevisedReprint: 2005 Third Revised Edition (multicolour) : 2006 Revised Reprint : 2007 @Copyright reserued by Dn U. Satyanarayana. Publishing rights and Printing rights reserved by the Publisher. Marketing rights, Distributing rights & Selling rights reserued by the Publisher. All rights reserved. No part of this publication may be reproduced or transmifted in any form or by any means, electronic, mechanical, photo-copying, recording or any informatign storage and retrieval system, without the prior wiitten permission of the Publisher. Exclusive rights reserued by the Publisher for publishing, printing, sale, marketing, distribution, expoft and translation of this book for all editions and reprints thereof. Cover Design Depicts the universal energy currency of the living world-ATP, predominantly synthesized by the mitochondria ol the cell (the functional unit of life), in comparison with the intemational currencies--$, t, €, Rs, Y. Publisher Typesetter Printer ArunabhaSen BOOKS AND ALLIED (P) Lro. 8/1 ChintamoniDas Lane, Kolkata700009 BOOKS AND ALLIED (P) Lro. 8/1 ChintamoniDas Lane, Kolkata700009 SWAPNA PRINTINGWORKS (P) Lro. 52 Raja RammohanRoy Sarani,Kolkata700009 ShyamalBhattacharyaProject Supervisor : tsBN Bt-8?!1q-80-t Price: Rs.575.00lRupeesFivehundredandSeventy{ive)only US$12.00only AuthorsSponsored& Supportedby : UIBFALAAUTFIOR-PUBTISHERINTERTINKS D.No.: 48-16-10,NagarjunaNagar,MahanaduRoad,Vrjayawada-520008(A"Pl
  • Prefaceto the Third Edition Theresponseto the first andthe secondeditionsof my book'Biochemistry'(reprintedseveraltimesin just 6 years)from the studentsandteachersis simplyoverwhelming.I wasfloodedwith highlyappreciative lettersfrom all cornersof Indiaandabroad!Thisgivesme immensesatisfactionandencouragemLntin this academicventure. I havecorrespondedwith manybiochernistryteachers,invitingtheir commentsandopinionsfor further improvingthe book.Mostof them havebeenkind enoughto offerconstructivesuggestions.I alsovisited severalcollegesandhadpersonalinteractionwith facultymembersandstudents.Theseexercises,spreadover the past 6 years,have helpedme to get direct feedbackon my book, besidesrealisingthe additional requirementsof students. I havegreatpleasurein presentingthe third editionof my bookwith severalunique/novelfeatures,some high-lightsof which are listedbelow. . A thoroughrevisionandupdatingof eachchapterwith latestadvances- . Multicolouredillustrationsfor a betterunderstandingof chemicalstructuresandbiochemicalreactions. . Increasein the font sizeof the text for morepleasantandcomfortablereading. o Incorporationof a newSectionon MolecularBiologyandBiotechnology. . Additionof ten new chapters-humangenomeproject,genetherapy,bioinformatics,free radicalsand antioxidants,tissueproteinsandbodyfluids,environmentalbiochemistry,genetics,immunologyetc. . An improvedorientationand treatmentof humanbiochemistryin healthanddisease. . Additionof practicalbiochemistryandclinicalbiochemistrylaboratoryin the appendix. It is true that I representa selectedgroupof individualsauthoringbooks,havingsometime at disposal, besideshardwork,determinationanddedication.I considermyselfan eternallearneranda regularstudent of biochemistry.However,it is beyondmy capabilityto keeptrackof theevergrowingadvancesin biochemistry dueto the exponentialSrowthof the subject.And this makesme nervous,wheneverI think of revisingthe book.I honestlyadmitthat I haveto dependon maturereadersfor subsequenteditionsof this book. AN INVITATION TO READERS It is not all the time possiblefor me to meetthe readersindividuallyandgettheir feedback,despitemy ferventwish.Of course,I dowrite to somepeoplepersonaliyseekingtheir opinions.However,I wishto have the commentsandsuggestionsof eachoneof the readersof my book.I sincerelyinvitethe readersto feelfree andwrite to me expressingtheir frank opinions,criticalcommentsandconstructivesuggestions. DT.U. SATYANARAYANA trl
  • I owea deepdebtof gratitudeto my parents,the lateSri U.VenkataSubbaiah,andSmt. Vajramma,for cultivatingin me the habitof earlyrising.Thewriting of this bookwouldneverhavebeenpossiblewithout this healthyhabit.I am gratefulto Dr. B. S.NarasingaRao(formerDirector,NationalInstituteof Nutrition, Hyderabad)for discipliningmy professionallife, andto my eldestbrother Dr. U. Gudaru(formerProfessorof PowerSystems,WalchandCollegeof Engineering,Sangli)for discipliningmy personallife. My elder son, U. Chakrapani(MBBS)deservesa specialplacein this book. He madea significant contributionat everystageof its preparation-writing, verification,proof-readingandwhat not. I hadthe rare privilegeof teachingmy sonashehappenedto bea studentof our college.Anda majorpartof this bookwas writtenwhilehewaslearningbiochemistry.Thus,hewasthe firstpersonto learnthesubjectof biochemistry from my handwrittenmanuscript.Thestudent-teacherrelation(ratherthan the father-son)hashelpedme in receivinSlconstantfeedbackfrom him and restructurethe book in a way an undergraduatestudentwould expecta biochemistrytextbookto be. Next,I thankDr. G.PitcheswaraRao(formerProfessorof Anatomy,SMC,Vijayawada)for his constructive criticism and advice,and Dr. B. Sivakumar(Director,NationalInstituteof Nutrition, Hyderabad)for his helpful sugi5lestionson the microfigures.I am gratefulto my nephew,Mr. U. SrinivasaRao,for helping me in drawingsomefigures. Last but not least,I thank my wife Krishna Kumari and my youngerson,Amrutpani,without whose cooperationand encouragementthis book could never have beenwritten. The manuscriptwas carefully nurturedlike a newborn babyandthe bookhasnow becomea full-pledgedmemberof our family. ACKNOWLEDGEMENTSTO THE THIRD EDITION I amindebtedto a largenumberof friends,pen-friendsandstudentswhohelpedmeto reviseandimprove the qualityof this book.I haveindividuallyandpersonallythankedall of them (whonumbera fewhundreds!). I onceagainexpressmy gratitudeto them. I thank my friend and colleague,Mr. M.S.T.JaganMohan,who has helpedme with his frequent interactionsto improvethe book,andmakeit morestudent-friendly.I wouldlike to placeon recordmy deep senseof appreciationto my post-graduate(M.D.)students,Dr. (Mrs.)U.B.VijayaLakshmiandDr. (Mrs.)Vidya DesaiSripad,whoseperiodicalacademicinteractionandfeedbackhavecontributedto the improvementof the biomedicaVclinicalaspectsin somechapters.I acknowledgethe helpof my friend,Dr. P.Ramanujam(Reader in English,AndhraLoyolaCollege,Vijayawada)for his helpandencouragementin revisingthe book. I expressmy gratitudeto Mr. ArunabhaSen, Director,Books & Allied (P) Ltd. Kolkata,for his wholeheartedsupportand constantencouragementin revisingthe bookin multicolour,and takingall the painsto bring it out to my satisfaction.I thank Mr. ShyamalBhattacharyafor his excellentpage-makingand graphics-workin the book.I am indebtedto Mr. PrasenjitHalderfor the coverdesignof this book. I thank my wife, Krishna Kumari, and my younger son, Amrutpani, for their constantsupport and encouragement.I am grateful to UppalaAuthor-PublisherInterlinks, Vijayawada,for sponsoringand supportingme to bring out this edition. Iiii] DT.U. SAIYANARAYANA
  • Biochemistry The term Biochemistrywas introducedby Carl Neubergin 1903.Biochemistrybroadlydealswith the chemistrvof life and living processes.Thereis no exaggerationin the statement,'Thescopeof biochemistrg is asuastaslilb itself!' Everyaspectof life-birth,growth,reproduction,aginganddeath,involvesbiochemistry. For that matter,everymovementof life is packedwith hundredsof biochemicalreactions.Biochemistryis the mostrapidlydevelopingandmostinnovativesubjectin medicine.Thisbecomesevidentfromthe factthat over the years,the major shareof NobelPrizesearmarkedfor Medicineand Physiologyhasgoneto researchers engagedir: biochemistry. The disciplineof biochemistryservesas a torch light to trace the intricate complexicitiesof biology, besidesunravellingthe chemicalmysteriesof life.Biochemicalresearchhasamplydemonstratedthat all living thingsarecloselyrelatedat the molecularlevel.Thusbiochemistryis the subjectof unity in the diversified living kingdom. Advancesin biochemistryhavetremendousimpacton humanwelfare,andhavelargelybenefitedmankind and their living styles.Theseincludethe applicationof biochernistryin the laboratoryfor the diagnosisof diseases.the products(insulin,interferon,€rowthhormoneetc.)obtainedfrom geneticengiineering,andthe possibleuseof genetherapyin the nearfuture. 0rganizationof the Book This texthook,comprising43 chapters,is orgianizedinto serrensecl:ionsin the heirarchicalorder of learninSbiochemistry. . SectionI dealswith the chemicalconstituentsof life-carbohydrates,lipids,proteinsandaminoacids, nucleicacidsandenzymes. . SectionII physiologicalchemistryincludesdigestionandahsorption,plasmaproteins,hemoglobinand prophyrins,andbiologicaloxidation. . SectionIII incorporatesall the metabolisms(carbohydrates,lipids,aminoacids,nucleotides,minerals) . Section[V covershormones,organfunctiontests,water,electrolyteandacid-basebalance,tissueproteins andtrodi'fluids,andnutrition. . SectionV is exclusivelydevotedto molecularbiologyandbiotechnology(DNA-replication,recombination, ar"lnrepair,transcriptionandtranslation,regulationof geneexpression,recombinantDNAandbiotechnology) . SectionVI givesrelevantinformation on current topics such a^shuman genomeproject,genetherapy, bioirrtormatics,prostaglandins,diabetes,cancer,AIDSetc. . Section VII dealswith the basic aspectsfor learning and understandingbiochemistry (bioorganic chenristry',hiophysicalchemistrytoolsof biochemistry,genetics,immunology). Each chapterin this book is carefully craftedwith colour illustrations, headingsand subheadingsto facilitatequickunderstanding.Theimportantapplicationsof biochemistryto humanhealthanddiseaseareput togetherasbiomedical/clinicalconcepts.Iconsare usedat appropriateplacesto serveas 'landmarks'. The origins of biochemicalwords, confusablesin biochemistry,practicalbiochemistryand clinical biochemistrylaboratory,givenin the appendixare novelfeatures. Thebriokis so organizedasto equipthe readerswith a comprehensiveknowledgeof biochemistry. Iiu]
  • Gontents SECTION ONE ChemicalConstituentsof Life 1 > Biomoleculesandthecell 2 > Carbohydrates 3 > Lioids 4 > Proteinsandaminoacids 5 > Nucleicacidsandnucleotides 6 > Enzymes 7 > Vitamins SECTION TWO PhysiologicalBiochemistry B > Digestionandabsorption 9 > Plasmaoroteins 10 > Hemoglobinandporphyrins 11 > Biologicaloxidation SECTION THBEE q3 > Metabolismofcarbohydrates *4 > Metabolismoflioids F-, Metabolismofaminoacids 16 > Int6grationofmetabolism 17 > Metabolismofnucleotides 1B > Mineralmetabolism SECTION FOUR ClinicalBiochemistrvand Nutrition 19 > Hormones 20 > Organfunctiontests 21 > Water,electrolyteand acid-basebqlance 22 > Tissueproteinsandbodyfluids 23 > Nutrition- SECTION FIVE MolecularBiologyand Biotechnology 24 > DNA-replication,recombinationandrepair523 25 > Transcriotionandtranslation 542 26 > Regulationofgeneexpression 566 27 b RecombinantDNAandbiotechnology578 sEcTtcN stx Current Topics 28 > Humangenomeproject 619 29 > Genetherapy 625 30 F Bioinformatics 634 31 p 'lvletabolismofxenobiotics(detoxification)638 32 >' Prostaglandinsandrelatedcompounds644 33 > Biologicalmembranesandtransport 650 34 b Freeradicalsandantioxidants 655 35 > Environmentalbiochemistry 662 36 l" Insulin,glucosehomeostasis, 3 9 28 43 69 85 176 165 182 196 221 anddiabetesmellitus Cancer 669 58s37> 38> Acquiredimmunodeficiency syndrome(AIDS) 695 241 244 285 330. 380 387 403 427 453 SECTION SEVEN Basicsto LearnBiochemistrv 39 > Introductiontobioorganicchemistry 40 > Overviewofbiophysicalchemistry 41 > Toolsofbiochemistrv 42 > lmmunology 43 > Genetics APPENDICES AnswerstoSelf-assessmenlExercises I Abbreviationsusedinthisbook' ll Greekalphabets lll Originsolimportantbiochemicalwords lV Commonconfusablesinbiochemistry V Practicalbiochemistry-principles Vl Clinicalbiochemistrylaboratory INEEX 703 708 719 732 737 745 751 756 tJt 760 764 770 773 468 487 502
  • fi Protuinsand Amino acids 4: Nucleicacidsand Nucleotides 69
  • BflomnoXeeutrssaildthsCelll -l- hu living matter is composedof mainly six I elements-carbon, hydrogen, oxygenl nitrogen, phosphorus and sulfur. Theseelements togetherconstituteabout 90% of the dry weight of the human body. Severalother functionally importantelementsare also found in the cells. Theseinclude Ca, K, Na, Cl, Mg, Fe,Cu, Co, l, Zn, F, Mo and Se. earbon-a unique element of life Carbonis the most predominantand versatile elementof life. lt possessesa unique propertyto form infinite number of compounds. This is attributedto the ability of carbon to form stable covalentbonds and C-C chains of unlimited length. lt is estimated that about 90% of compounds found in living system invariably contain carbon. Ghemical molecules of li#e Life is composed of lifeless chemical molecules. A single cell of the bacterium, Escherichiacoli containsabout 6.000 different organiccompounds.lt is believedthat man may contain about 100,000 different types of moleculesalthough only a few of them have been characterized. Sornpiex *riomoleeules The organiccompoundssuchasamino acids, nucleotidesand monosaccharidesserve as the monomeric unitsor building blocksof complex biomolecules-proteins,nucleicacids(DNA and RNA) and polysaccharides,respectively.The important biomolecules(macromolecules)with their respective building blocks and major functions are given in Table 1.1. As regards lipids, it may be noted that they are not biopolymers in a strict sense,but majority of them contain fatty acids. Structural heirarehy off asn organisnl The macromolecules(proteins,Iipids,nucleic acidsand polysaccharides)form supramolecular assemblies(e.g. membranes)which in turn organize into organelles,cells, tissues,organs and finally the whole organism. 3
  • BIOCHEMISTFIY Biomolecule Buildingblock (repeatingunit) Major functions 1. Protein Aminoacids 2. Deoxyribonucleicacid(DNA) Deoxyribonucleotides Ribonucleotides3. Ribonucleicacid(RNA) 4. Polysaccharide(glycogen)Monosaccharides(glucose) Fundamentalbasisofstructureand functionofcell(staticanddynamicfunctions). fl_eq_o_sitoryo.l.!9199iFryi{9l1llgt Essentiallyrequiredlorproteinbiosynthesis. Storageformofenergytomeetshortterm demands. 5. Lipid Fattyacids,glycerol Storagetormofenergytomeetlongterm demands;structuralcomponentsofmembranes. Chem*ca! composition of man The chemicalcompositionof a normal man, weighing 65 kg, is given in Table 1.2.Water is the solventof life and contributesto more than 60"h of the weight. This is followed by protein (mostlyin muscle)and lipid (mostlyin adipose tissue).The carbohydratecontent is rather low which is in the form of glycogen. The cell is the structuraland functional unit of life. ft may be also regardedas the basic unit of hiological activity. The concept of cell originated from the contributionsof Schleidenand Schwann(1838). However, it was only after 1940, the complexitiesof cell structurewere exposed. Constituent Percent(7") Weight (kg) Prokaryotic and eukaryotic cells The cells of the living kingdom may be divided into two categories 1. Prokaryotes(Creek: pro - before;karyon- nucleus)lacka well definednucleusand possess relatively simple structure.These include the variousbacteria. 2. Eukaryotes(Greek: eu-true; karyon- nucleus)possessa well definednucleusand are more complex in their structureand function. The higher organisms(animalsand plants)are composedof eukaryoticcells. A comparisonof the characteristicsbetween prokaryotesand eukaryotesis listedin Table 1.3. The human body is composedof about 1014 cells.There are about 250 typesof specialized cel{s in- the human body'G.g. erythrocytes, nerve-cells, muscle cells, B cells of pancreas. An eukaryoticcell is generally10 to 100 pm in diameter. A diagrammatic representation of a typical rat liver cell is depicted in Fig.I.t. The plant cell differsfrom an animalcell by possessinga rigid cell wall (mostlycomposedof cellulose)and chloroplasts.The latter are the sitesof photosynthesis. Water Protein Lipid Carbohydrate Minerals 61.6 17.0 13.8 6.1 40 11 I '| 4
  • Chapter 1 : BIOMOLECULESAND THE CELL Characteristic Prokaryotic cell Eukaryoticcell 1. Size Small(generally1-10pm) Large(generally10-100pm) 2. Cellmembrane Cellisenvelopedbyaflexibleplasmamembrane Distinctorganellesarefound (e.9.mitochondria,nucleus,lysosomes) 3. Sub-cellular organelles 4, Nucleus Notwelldefined;DNAisfound asnucleoid,histonesareabsent Nucleusiswelldefined,surroundedbya membrane:DNAisassociatedwithhistones 5. Energymetabolism Mitochondriaabsent,enzymesof energymetabolismboundto Enzymesolenergymetabolismarelocated inmitochondria membrane 6. Celldivision 7. Cytoplasm Usuallyfissionandnomitosis Mitosis 0rganellesandcytoskeleton absent Containsorganellesandcytoskeleton (anetworkoftubulesandfilaments) The cell consistsof well definedsubcellular organelles,envelopedby a plasma membrane. By differential centrifugation of tissue homogenate, it is possible to isolate each cellular organelle in a relatively pure form (Refer Chapter 41). The distribution of major enzymes and metabolic pathways in different cellular organelles is given in the chapter on enzymes (Refer Fig.6.6). The subcellular organellesare briefly describedin the following pages. Nucleus Nucleus is the largest cellular organelle, surroundedbv a double membrane nuclear envelope.The outer membraneis continuous with the membranesof endoplasmicreticulum. At certainintervals,the two nuclearmembranes have nuclearporeswith a diameterof about 90 nm. Theseporespermit the free passageof the products synthesizedin the nucleus into the surrounding cytoplasm. Roughendoplasmicreticulum Golgiapparatus Lysosome Mitochondrion Plasmamembrane Vacuole Ribosomes Peroxisome Cytoskeleton Cytosol Coatedpits Ftg. 1.1: Diagrammaticrepresentationof a nt liverell.
  • BIOCHEMISTF|Y Nucleus contains DNA, the repository of genetic information. Eukaryotic DNA is associatedwith basic protein (histones)in the ratio of 1 : 1, to form nucleosomes.An assembly of nucleosomesconstituteschromatin fibres of chromosomes(Creek'.chroma - colour; soma- body). Thus, a single human chromosomeis comoosedof abouta million nucleosomes.The number of chromosomes is a characteristic feature of the species. Humans have 46 chromosomes,compactlypackedin the nucleus. The nucleusof the eukaryoticcell containsa dense bodv known as nucleolus.lt is rich in RNA, particularlythe ribosomal RNA which entersthe cytosolthrough nuclearpores. The ground materialof the nucleus is often referredto as nucleoplasm.lt is rich in enzymes such as DNA polymerases and RNA polymerases.To the surpriseof biochemists,the enzymes of glycolysis,citric acid cycle and hexose monophosphateshunt have also been detectedin the nucleoplasm. Mitochondria The mitochondria (Creek'. mitos- thread; chondros- granule) are the centres for the cellularrespirationand energymetabolism.They are regarded as the power housesof the cell with variablesize and shape.Mitochondriaare rod-like or filamentousbodies, usuallv with dimensions of 1.0 x 3 pm. About 2,0O0 mitochondria,occupyingabout 1/5thof the total cell volume,are presentin a typicalcell. The mitochondriaare comoosedof a double membrane system. The outer membrane is smooth and completelyenvelopsthe organelle. The inner membrane is folded to form cristae (Latin- crests)which occupy a larger surface area. The internal chamber of mitochondriais referred to as matrix or mitosol. The componentsof electron transportchain and oxidative phosphorylation (flavoprotein, cytochromesb, c1, C, a and a3 and coupling factors)are buried in the inner mitochondrial membrane.The matrixcontainsseveralenzvmes concerned with the energy metabolism of carbohydrates,lipidsandaminoacids(e.g.,citric acid cycle, p-oxidation).The matrix enzymes also parlicipate in the synthesisof heme and urea. Mitochondria are the principal producers of ATP in the aerobic cells. ATP, the energy currency,generatedin mitochondriais exported to all partsof the cell to provideenergyfor the cellularwork. The mitochondrialmatrixcontainsa circular double stranded DNA (mtDNA), RNA and ribosomes.Thus,the mitochondriaareequipped with an independent protein synthesizing machinery.It is estimatedthat about 10% of the mitochondrial oroteins are produced in the mitochondria. The structureand functionsof mitochondria closely resemble prokaryotic cells. lt is hypothesizedthat mitochondria have evolved from aerobicbacteria.Further,it is believedthat duringevolution,the aerobicbacteriadeveloped a symbiotic relationship with primordial anaerobiceukaryoticcellsthat ultimatelyled to the arrival of aerobiceukaryotes. Endoplasmic reticulum The network of membraneenclosedspaces that extends throughout the cytoplasm constitutesendoplasmicreticulum(ER).Someof these thread-like structuresextend from the nuclearporesto the plasmamembrane. A large portion of the ER is studded with ribosomesto give a granularappearancewhich is referred ro as rough endoplasmic reticulum. Ribosomes are the factories of protein biosynthesis. During the process of cell fractionation,roughERisdisruptedto form small vesiclesknown as microsomes.It may be noted that microsomesas such do not occur in the cell. The smoothendoplasmicreticulumdoes not containribosomes.lt is involvedin the synthesis of lipids (triacylglycerols,phospholipids,sterols) and metabolismof drugs,besidessupplyingCa'?. for the cellularfunctions. Golgi apparats,r$ Eukaryoticcells contain a unique clusterof membrane vesicles known as dictyosomes
  • Chapter 1 : BIOMOLECULESAND THE CELL which, in turn, constituteColgi apparatus(or Colgi complex).The newly synthesizedproteins are handed over to the Colgi apparatuswhich catalysethe addition of carbohydrates,lipids or sulfatemoietiesto the proteins.Thesechemical modificationsare necessaryfor the transportof proteinsacrossthe plasmamembrane. Certainproteinsand enzymesareenclosedin membrane vesicles of Colgi apparatusand secreted from the cell after the appropriate signals.The digestiveenzymesof pancreasare oroducedin this fashion. Colgi apparatusare also involved in the membrane synthesis, particularly for the formation of intracellular organelles (e.g. peroxisomes,lysosomes). Lysosornes Lysosomesare sphericalvesiclesenveloped by a singlemembrane.Lysosomesare regarded as the digestivetract of the cell, sincethey are actively involved in digestion of cellular substances-namely proteins, lipids, carbo- hydratesand nucleic acids.Lysosomalenzymes are categorizedas hydrolases.Theseinclude the following enzymes(with substratein brackets) a-C lucosidase(glycogen) Cathepsins(proteins) Lipases(lipids) Ribonucleases(RNA) The pH of the lysosomalmatrixis moreacidic (pH< 5) than the cytosol (pH-7) and this facilitatesthe degradationof differentcompounds. The lysosomal enzymes are responsiblefor maintaining the cellular compounds in a dynamic stafe, by their degradationand recycling.The degradedproductsleavethe lysosomes,usually by diffusion, for reutilization by the cell. Sometimes,however,certain residualproducts, rich in lipidsand proteins,collectivelyknown as Iipofuscinaccumulatein the cell. Lipofuscinis the agepigmentor wear and tearpigmentwhich has been implicatedin ageingprocess. The digestiveenzymesof cellularcompounds are confinedto the lvsosomesin the bestinterest of the cell. Escapeof theseenzymesinto cytosol will destroythe functionalmacromoleculesof tne cell and result in many complications.The occurrence of several diseases(e.g. arthritis, musclediseases,allergicdisorders)hasbeenpartly attributedto the releaseof lysosomalenzymes. Feroxisomes Peroxisomes,also known as microbodies, are single membranecellularorganelles.They are spherical or oval in shape and contain the enzyme catalase.Catalaseprotectsthe cell from the toxic effectsof HrO, by converting it to HrO and Or. Peroxisomesare also involved in tne oxidation of long chain fatty acids (> C,s),and synthesisof plasmalogensand glycolipids.Plants contain glyoxysomes, a specialized type of BTOMED|eAL/ CLINICAL COIUCEPTS A liuing cell is a true representotiueof life with its own organizotionand specialized lunctions. Accumulotion oJ lipofuscin,a pigment rich in lipids and proteins, in the cell hasbeen implicated in ogeing process. Leokageof lysosomalenzymesinto the cell degrodesseuerolfunctional macromolecules and this may leod to certain disorders (e.9. arthritis). rq Zellweger syndrome is a rare diseose characterized by the absence of functional peroxisomes.
  • E}IOCHEMISTF|Y peroxisomes, which are involved in the glyoxylate pathway. Peroxisome biogenesisdisorders (PBDs), are a Broup of rare diseasesinvolving the enzyme activities of peroxisomes. The biochemical abnormalitiesassociatedwith PBDs incluoe increasedlevelsof very long chain fatty acids (C2aand C26)and decreasedconcentrationsof plasmalogens.The most severeform of PBDsis Zellweger syndrome, a condition characterized by the absenceof functional peroxisomes.The victimsof this diseasemav die within one vear after birth. {iytosol and cytoskeleton The cellular matrix is collectively referredto as cytosol. Cytosol is basicallya compartment containing several enzymes/ metabolites and saltsin an aqueousgel like medium.More recent studies however, indicate that the cytoplasm actuallycontainsa complex network of protein filaments, spread throughout, that constitutes cytoskeleton.The cytoplasmicfilamentsare of three types- microtubules, actin filaments and intermediatefilaments.The filamentswhich are polymers of proteins are responsiblefor the structure,shapeand organizationof the cell. INTEGRATIOI{ OF CELLULAR FUNCTIONS The eukaryoticcells performa wide rangeof complex reactionsfunctionsto maintaintissues, and for the ultimatewell-beingof the whole organism. For this purpose, the various intracellularprocessesand biochemicalreactions are tightly controlledand integrated.Divisionof a cell intotwo daughtercellsis goodexampleof the orderlyoccurrenceof an integratedseriesof cellularreactions. Apoptosisis the programmedcell death or cell suicide. This occurs when the cell has fulfilled its biologicalfunctions.Apoptosismay be regardedas a natural cell deathand it differs from the cell death caused by injury due to radiation,anoxiaetc. Programmedcell death is a highly regulatedprocess. 1. 2. 3. Life is composed ol lifeless chemical molecules. The complex biomolecules, proteins, nucleic ocids (DNA and RNA), polysaccharidesand lipids are formed by the monomeric units amino acids,nucleotides,monosaccharidesand fotty acids,respectluely. The cell is the structuroland functional unit of life. The eukoryoticcell consisfsof well det'inedsubcellulororganelles,enuelopedin a plasma membrane. The nucleus contoinsDNA, the repositoryol genetic int'ormation.DNA, in association with proteins (histones),forms nucleosomeswhich,in turn, make up the chromosomes. The mitochondria qre the centresfor energymetobolism. Theyare the principalproducers of ATP which is exported to all parts of the cell to ptouide energylor cellular work. Endoplosmic reticulum (ER) ts the network of membrane enclosed spocesthat extends throughout the cytoplosm. ER studded with ribosomes, the factorles of protein biosynfhesis, ts relerred to as rough ER. Golgi opparatus sre a cluster of membrane uesiclesto uthich the newlg synthesizedproteins are handed ouer for t'urther processing ond export. Lysosomesare the digestiue bodiesol the cell, actiuely involued in the degradotion of cellular compounds. Peroxisomescontoln the enzymecatalosethat protects the cell lrom the toxic elfects of HrOr. The cellular ground motrix is referred to as cytosol which, in fact, is composed of a network ot' protein t'ilaments, the cytoskeleton. Theeukaryoticcellsperform a widerangeof complex lunctionsin a well coordinatedand integrated fashion. Apoptosis is the processol programmed cell death or cell suicide. 5. 6. 7.
  • 1^ arbohydratesare the most abundantorganic - molecules in nature. They are primarily composedof the elementscarbon, hydrogen and oxygen.The name carbohydrateliterallymeans 'hydratesof carbon'.Someof the carbohydrates possessthe empiricalformula (C.H2O)nwhere n 3 3, satisfyingthat thesecarbohydratesare in fact carbonhydrates.However,thereare several non-carbohydratecompounds(e.g. acetic acid, C2HaO2;lacticacid,C3H6O3)which alsoappear as hydratesof carbon. Further, some of the genuine carbohydrates (e.g. rhamnohexose, C6H12O5ideoxyribose,C5H16Oa)do not satisfy the generalformula.Hencecarbohydratescannot be alwaysconsideredas hydratesof carbon. Carbohydrates may be defined as polyhydroxyaldehydes or ketones or compounds which produce them on hydrolysis. The term 'sugar' is applied to carbohydratessoluble in water and sweet to taste. #-ur*c;tEerEsof earbohydrates Carbohydratesparticipatein a wide rangeof functions 1. Theyarethe mostabundantdietarysource of energy (a Cal/S)for all organisms. 2. Carbohydratesare precursorsfor many organic compounds(fats,amino acids). 3. Carbohydrates(asglycoproteinsand glyco- lipids) participate in the structure of cell membraneand cellular functionssuch as cell growth, adhesionand fertilization. 4. They are structuralcomponentsof many organisms.Theseincludethe fiber (cellulose)of plants,exoskeletonof some insectsand the cell wall of microorganisms. 5. Carbohydratesalso serve as the storage form of energy(glycogen)to meetthe immediate energydemandsof the body. CLASSIFICATION OF GARBOHYDRATES Carbohydrates are often referred to as saccharides (Greek: sakcharon-sugar).They are broadlyclassifiedinto three major groups- monosaccharides, oligosaccharides and polysaccharides.This categorizationis basedon
  • t0 BIOCHEMISTRY Monosaccharides(empiricalformula) AIdose Ketose Trioses(CgHoOg) Telroses(C+HoO+) Pentoses(CsHroOs) Hexoses(CoHrzOo) Heptoses(CzHr+Oz) Glyceraldehyde Erythrose Ribose Glucose Glucoheptose Dihydroxyacetone Erythrulose Ribulose Fructose Sedoheptulose the number of sugar units. Mono- and oligo- saccharidesare sweet to taste, crystalline in characterand soluble in water, hence thev are commonly known as sugars. FJtonosaccharides Monosaccharides(Greek: mono-one)are the simplestgroup of carbohydratesand are often referred to as simple sugars.They have the generalformula Cn(H20)n,and they cannot be further hydrolysed.The monosaccharidesare divided into differentcategories,based on the functionalgroupandthe numberof carbonatoms Aldoses : When the functional group in IH monosaccharidesis an aldehydel-C:oi, ,h"u are known as aldoses e.g. glyceraldehyde, glucose. Ketoses: When the functionalgroup is a keto lt -C:O.l group, they are referredto as ketoses e.g. dihydroxyacetone,fructose. Basedon the number of carbon atoms,the monosaccharidesare regarded as trioses (3C), tetroses (4C), pentoses (5C), hexoses (6C) and heptoses(7C).Thesetermsalongwith functional groupsare usedwhile namingmonosaccharides. For instance, glucose is an aldohexose while fructose is a ketohexose(Table 2,1). Thecommonmonosaccharidesand disaccha- rides of biological importanceare given in the Table 2.2. SSlgosaccharides Oligosaccharides(Creek: oligo-few) contain 2-1O monosaccharidemolecules which are liberatedon hydrolysis.Basedon the numberof monosaccharide units present, the oligo- saccharides are further subdivided to disaccharides,trisaccharidesetc. Polysace harides Polysacchari6ls(Creek:poly-many)are poly- mers of mondficcharide units with high mole- cular weight (up to a million).They are usually tasteless(non-sugars)and form colloids with water. The polysaccharidesare of two types- homopolysaccharidesand heteropolysaccharides. Stereoisomerismis an importantcharacterof monosaccharides. Stereoisomers are the compounds that have the same structural formulaebut differ in their spatialconfiguration. A carbon is said to be asymmetric when it is attached to four different atoms or groups. Ihe number of asymmetric carbon atoms (n) determines the possible isomers of a given compound which is equal to 2n. Clucose contains4 asymmetriccarbons,and thus has 16 tsomers. Glyeeraldehyde -tfu e ref erqlrt*e cff rb$hyd$'er'&€3 Clyceraldehyde(triose)is the simplestmono- saccharidewith one asymmetriccarbonatom. lt existsastwo stereoisomersand hasbeenchosen as the referencecarbohydrateio representthe structureof all other carbohvdrates.
  • Ghapter 2 : CARBOHYDRATES 11 Trioses Glyceraldehyde Dihydroxyacetone Tetroses D-Erythrose Foundincellsasphosphate Foundincellsasphosphate i Widespread I Widespreadasaconstituentof I RNAandnucleotides i AsaconstituentofDNA : Producedduringmetabolism i Asaconstituentofglycoproteins i anogums i ls anintermediateinuronicacidpathway i Heartmuscle i --. --. -- --.. ---.. -.. -. --. Asaconstituentolpolysaccharides (starch,glycogen,cellulose)and disaccharides(maltose,lactose, sucrose).Alsofoundinfruits Asaconstituentoflactose (milksugar) Foundinplantpolysaccharides andanimalglycoproteins Fruitsandhoney,asaconstituent ofsucroseandinulin Foundinolants i Glyceraldehyde3-phosphateisanintermediate i inglycolysis i ttst-pnosphateisanintermediateinglycolysis ----t------..-..-.--...--- ForthestructureofRNAandnucleotide coenzymes(ATP,NAD+,NADP+) ForthestructureolDNA Itisanimportantmetaboliteinhexose monophosphateshunt Involvedinthefunctionofglycoproteins Excretedinurineinessenlialpentosuria Asaconstituentollvxollavinofheartmuscle The'sugarfuel'oflife;excretedinurinein diabetes.Structuralunitofcelluloseinplants Convertedtoglucose,failureleadsto galactosemia Forthestructureofpolysaccharides Itsphosphatesareintermediatesofglycolysis Its7-phosphateisanintermediateinhexose monophosphateshunt,andinphotosynthesis Pentoses D-Ribose D-Deoxyribose D-Ribulose D-Xylose L-Xylulose D-Lyxose Hexoses D-Glucose D-Galactose D-Mannose D-Fructose Heptoses D-Sedoheptulose Disaccharides Occurrence Biochemical importance Sucrose Lactose Asaconstituentofcanesugarand beetsugar,pineapple Milksugar Productofstarchhydrolysis, occursingerminatingseeds Mostcommonlyusedtablesugarsupplying calories Exclusivecarbohydratesourcetobreastfed infants.Lactasedeficiency(lactoseintolerance) leadstodianheaandflatulence Animportantintermediateinlhedigestionof starch Maltose
  • 12 E}IOCHEMISTFIY H-C:O I H-C-OH cH2oH D-Glyceraldehyde H-C:O HO-C-H cH2oH L-Glyceraldehyde H-C:O I HO-C-H H-C-OH I HO-C-H HO-C-H cH2oH L-Glucose Fig.2.1 : DandL- formsof glucosecomparedwith D and L- glyceraldehydes (the reference carbohydrate). D" and L-isomers The D and L isomersare mirror imagesof each other. The spatialorientationof -H and -OH groups on the carbon atom (Cs for glucose)that is adjacentto the terminal primary alcohol carbon determineswhetherthe sugaris D- or L-isomer.lf the -OH group is on the right side,the sugaris of D-series,and if on the left side, it belongs to L-series.The structuresof D- and L-glucosebasedon the referencemono- saccharide, D- and L-glyceraldehyde (glycerose) are depicted in Fig.2.1. It may be noted that the naturallyoccurring monosaccharidesin the mammaliantissuesare mostlyof D-configuration.Theenzymemachinery of cells is specific to metaboliseD-seriesof monosaccharides. fn the medical practice, the term dextroseis used for glucosein solution. This is becauseof the dextrorotatorynature of glucose. Optlcal activity of sugars Optical activity is a characteristicfeature of compounds with asymmetric carbon atom. When a beam of polarized light is passed througha solutionof an optical isomer,it will be rotated either to the right or left. The term dextrorotatory (+) and levorotatory (-) are used to compoundsthat respectivelyrotatethe plane of polarizedlight to the right or to the left. An optical isomer may be designatedas D(+), D(-), L(+)and L(-) basedon its structural relation with glyceraldehyde.lt may be noted that the D- and L-configurationsof sugarsare primarily based on the structure of glyceraldehyde,the optical activitieshowever, may be different. Racemicmixture : lf D- and L-isomersare presentin equal concentration,it is known as racemicmixtureor DL mixture.Racemicmixture does not exhibit any optical activity, since the dextro- and levorotatorv activities cancel each other. Configuration of D-aldoses The configuration of possible D-aldoses startingfrom D-glyceraldehydeis depicted in Fig.2.2. This is a representation of Killiani- Fischersynthesis,by increasingthe chain length of an aldose,by one carbon at a time. Thus, startingwith an aldotriose(3C),aldotetroses(4C), aldopentoses(5C) and aldohexoses(6C) are formed. Of the 8 aldohexoses,glucose,mannose and galactoseare the most familiar. Among these, D-glucose is the only aldose mono- saccharidethat predominantlyoccurs in nature. Gonfiguration of D-ketoses Startingfrom dihydroxyacetone(triose),there are five keto-sugarswhich are physiologicallr important.Their structuresare given in Fig,2.3 Epimers ff two monosaccharides differ from eac- other in their configuration around a singk specificcarbon (otherthan anomeric)atom. L*ei are referred to as epimersto each orher '.Fig,21 For instance, glucoseand galactose are efilwl with regardto carbon 4 (Ca-epimers- -^:i 's they differ in the arrangementof -OH g.'ELcr Ca. Clucose and mannose are epi-'e--' q drl regardto carbon 2 (C2-epimers). The interconversionof epimers e - I r::r'e to galactose and vice versai s i - -^,'- a* H-C:O I H-C-OH I HO-C-H I H-C-OH I H-Q-OH I cHzoH D-Glucose
  • Ghapter 2 : CABBOHYDFATES 13 Aldotriose (3c) Aldotetroses (4c) cHo I HOCH I Aldo toses HOCH ) I HCOH I cH2oH D-Lyxoee cHo HOCH I I Aldo- HOCH HOCH hexoses HoCH noCH (6c) tl HCOH HCOH tt cHzoH cHzoH D-Galactose D-Talose 'l t- cHo HOCH I HCOH HCOH cH2oH D-Arabinose / JT cHo cHo HCOH HOCH rl HOCH HOCH tl HCOH HCOH tl HCOH HCOH ll cH2oH cH2oH D-Glucose D-Mannose HCOH I HCOH cH2oH D-Ribose / JT cHo cHo tl HCOH HOCH HCOH HCOH tl HCOH HCOH tl HCOH HCOH ll cH2oH cH2oH D-Allose D-Altrose cHo HCOH cHo I HCOH I HCOH I HOCH I HCOH cH2oH D-Gulose cHo I HCOH cHo I HOCH I HCOH I HOCH HCOH I cH2oH D-ldose cHo I HCOH I cH2oH D-Erythrose D.Threooe cHo I HCOH I HOCH I HCOH cH2oH D-Xylose I / / *+ Fig.2.2 : ThestructuralrelationshipbetweenD-aldosesshownin Fischerprojection. (TheconfigurationaroundC2(ed) distinguishesthemembersof eachpair). epimerization, and a group of enzymes- namely-epimerases catalysethis reaction. Enantiomers Enantiomers are a special type of stereoisomers that are mirror images of eachother. The two membersare designatedas D- and L-sugars.Enantiomersof glucose are depicted in Fig.2.5. Majority of the sugarsin the higher animals (includingman) are of D-type (Fig.2.5'1. The term diastereomersis used to represent the sfereoisomers that are not mirror imagesof one another. For a better understanding structure, let us consider the hemiacetals and hemiketals, producedwhen an aldehydeor a with alcohol. of glucose formation of respectively ketone reacts
  • 14 E}IOCHEMISTRY ?H2oH C:O I cH2oH Dlhydroxyacetone cH2oH I C:O I HOCH HCOH I cH2oH D-Xylulose cH20H I C:O HCOH HCOH I cH2oH D-Ribulose cH2oH I C:O HOCH I HCOH I HCOH I cH2oH D-Fructose cH2oH I C:O I HOCH I HCOH I HCOH I HCOH I cH2oH D-Sedoheptulose Fig.2.3 : Structuresof ketosesof physiologicalimportance. ,H nt-C.1^ + R2-oH l- Rr- LJ Aldefry<b Alcohol Hemiacetal The hydroxyl group of monosaccharidescan react with its own aldehydeor keto functional group to form hemiacetaland hemiketal.Thus, the aldehydegroup of glucoseat C1 reactswith alcohol group at C5 to form two typesof cyclic hemiacetalsnamely a and B, as depicted in Fig.2.6. The configuration of glucose is conveniently represented either by Fischer formulaeor by Haworth projectionformulae. Fyranose and furanose structures Haworth projectionformulaeare depictedby a six-memberedring pyranose(basedon pyran) or a five-memberedring furanose (based on furan).The cyclic formsof glucoseare known as a-D-glucopyranose and c-D-glucofuranose (Fig.2.V. Anomers-nrutarotation The a and p cyclic forms of D-glucose are known as anomers.Thev differ from each other in the configurationonly around C1 known as anomericcarbon(hemiacetalcarbon).In caseof o anomer,the -OH group held by anomeric carbon is on the opposite side of the group -CH2OH of sugarring. The reverseis true for B-anomer.The anomersdiffer in certainphysical and chemical properties. Mutarotation : The a and p anomers of glucose have different optical rotations. The specific optical rotation of a freshly prepared glucose(c anomer)solutionin water is +112.2o which gradually changes and attains an equilibriumwith a constantvalue of +52.7". ln the presenceof alkali, the decreasein optical rotation is rapid. The optical rotation of p-glucose is +18.7o. Mutarotation is defined as the change in the specific optical rotation representing the interconversion of u and p H-C:O I H-C-OH I HO-C-H I HO- C -H H-C-OH I cH2oH D-Galactose H-C:O I H-C-OH I HO-C-H I H .C-OH I H-C-OH I CHzOH D-Glucose H-C=O I HO-C-H I HO-C-H I H-C-OH I H-C-OH I cH2oH D-Mannose H I C:O I f{ c-oH I HC-C-H i-l- c-oH H-C-OH I t"1-c-H HO H O=C HO_C- H I H-C- Cl-i I HO-C-H I HO-C- Fl I H-C- ii I OH Fig.2,4: Structuresof epimers(glucoseand galactose are Co-epimerswhileglucoseand mannoseare C2-epimers). L-Glucose D-Glucose H9.2.5 : Enantiomers(mirrorimages)ofglucose.
  • t5 Ghapter 2 : CARB I cH20H o'D'Glucose (+ 112.2" fil H6?H o-D-GlucoPYranose 1 H-C:O I H-C-OH I HO-C-H I H-C-OH tc H-C-OH I cH2oH D-Glucose (aldehYdeform) l/A H6?H HOH D-Glucose (aldehydeform,acYclic) iHron ftD-Glucose (+18.7-) (B) HOH FD-GlucoPYranose cH20H forms of D'glucose to an equilihrium mixture' Mutarotationdepictedin Fig'2'6, is summartzeo below. cx-D-Clucose# Equilibriummixture# B-D-Clucose + 112.2" + 52.7" + 18.7" (Specificoptical rotation tctl2p0) The equilibrium mixture contains 63o/" p-anomer and 36"/ocl-anomer of glucose with Fig.2.7: Structurcofglucose-pyranose andfuranosetorms' HOH cr-D-GlucoPYranose cH20H t- H-C-OFi OH HOH cr-D-Glucofuranose 17oopen chainform. ln aqueoussolution'the p forrn 'i, more predominant due to its stable conformation.The cr and p formsof glucoseare interconvertiblewhich occurs through a linear form. The latter, as such, is present in a" insignificantquantitY. Mutarotation of fructose z Frur' exhibits mutarotation.ln case or pyranose ring (six-memberqd' furanose(five-membered)'o' is attained.And fruqt' rotation of -92)2. Ihe conv' to levor ':ut" :;r' on is kn, anome' in alkalir When gt. severalhours,
  • chapter 2 : CAFIBoHYDFATES 15 I cH2oH cr-D-Glucose (+ 112.2") 1 H-C=C) I H-C-OH I HO-C-H I H-C-OH l5 H-C-OH cH2oH D-Glucose (aldehydeform) HOH D-Glucose (aldehydeform,acyclic) forms of D-glucose to an equilibrium mixture. Mutarotationdepictedin Fi9.2.6, is summarized below. s-D-Clucose# Equilibriummircture# p-D-Glucose + 112.2" + 52.7" + 18.7o (Specificoptical rotation talf;) The equilibrium mixture contains 63"/" p-anomer and 36h cl-anomerof glucosewith cr-D-Glucopyranose 17oopen chain form. In aqueoussolution,the p form is more predominant due to its stable conformation.The s and p formsof glucoseare interconvertiblewhich occurs through a linear form. The latter, as such, is present in an insignificantquantity. Mutarotation of fructose : Fructose also exhibits mutarotation.ln case of fructose,the pyranose ring (six-membered)is converted to furanose(five-membered)ring,till an equilibrium is attained.And fructosehas a specificoptical rotationof -92" at equilibrium. The conversion of dextrorotatory (+) sucrose to levorotatory fructose is explained under inversionof sucrose(seelater in this chapter). REACTIONS OF MONOSACCHARIDES Tautomerization or enolization The processof shiftinga hydrogenatom from one carbon atom to anotherto produce enediols is known as tautomerization. Sugarspossessing anomericcarbon atom undergotautomerization in alkalinesolutions. When glucoseis kept in alkalinesolutionfor severalhours,it undergoesisomerizationto form HOH o-D-Glucopyranose pD-Glucopyranose Fig. 2.6 : Mutarotation of glucose representing a and p anomers (A) Fischer projections (B) Haworth projections. Fig.2.7 : Structureof glucose-pyranose and furanoseforms. 20H cH2oH H c-D-Glucofuranose
  • 16 BIOCHEMISTFIY H n-C-ot H-C:O ( I H- -OH HO-( HO-( R Enediol (common) Fig.2.8 : Formationof a commonenediolfrom glucose,fructoseandmannose {fr,f,o,F|F|lPffi:!lo.t|tfr,ft:PI:Is?Iboncolnmonstnftar:?l,l D-fructose and D-mannose. This reaction- known as the Lobry de Bruyn-von Ekenstein transformatiorr-results in the formation of a common intermediate-namely enediol--$or all the three sugars,as depicted in Fig.2.8. Theenediolsare highlyreactive,hencesugars in alkaline solution are powerful reducing agents. ft+r,.luleFr'.lgr!s.lFeFtlsF The sugarsare classifiedas reducingor non- reducing.The reducingpropertyis attributedto the free aldehyde or keto group of anomeric carbon. ln the laboratory, many testsare employed to identify the reducing action of sugars.These incfude Benedict's test, Fehling's test, Barfoed's tesf etc. The reduction is much more efficient in the alkaline medium than in the acid medium. The enediolforms(explainedabove)or sugars reduce cupric ions (Cu2+)of copper sulphate to cuprous ions (Cu+), which form a yellow precipitate of cuprous hydroxide or a red precipitate of cuprous oxide as shown next. t2H2O+ CueO{- 2Cu(OH) It may be noted that the reducing property of sugarscannothelp for a specificidentificationof any one sugar,since it is a generalreaction. 0xida*iern Depending on the oxidizing agent used, the terminal aldehyde (or keto) or the terminal alcoholor both the groupsmay be oxidized.For instance,considerglucose: 1. Oxidation of aldehydegroup (CHO ------> COOH) resultsin the formationof gluconicacid. 2. Oxidation of terminal alcohol group (CH2OH------+COOH) leadsto the production of glucuronicacid. Reduetion When treatedwith reducing agentssuch as sodiumamalgam,the aldehydeor keto groupof monosaccharideis reduced to corresponding alcohol, as indicatedby the generalformula : H H-C:O H-C-Ol-t I RR The important monosaccharidesand their correspondingalcoholsare given below. D-Glucose D-Galactose------+D-Dulcitol D-Mannose ------+D-Mannitol D-Fructose --) D-Mannitol+ D-Sorbitol D-Ribose -+ D-Ribitol Sorbitol and dulcitol when accumulate in tissuesin large amounts cause strong osmotic effectsfeadingto swelling of cells,and certain pathologicalconditions.e.g.cataract,peripheral neuropathy,nephropathy.Mannitol is usefulto reduce intracranialtensionbv forced diuresis.
  • Ghapter 2 : CAFIBOHYDRATES 17 H-C--O I H-C-OH I HO-C-H I H-C-OH I H-C-OH I cH2oH D-Glucose H-C:O I H-C:O I cH20H Hydrorymethylfurfural H-C:O I Formation of esters The alcoholic groups of monosaccharides may be esterified by non-enzymatic or enzymatic reactions. Esterificationof carbo- hydrate with phosphoric acid is a common reaction in metabolism.Glucose 6-phosphate and glucose 1-phosphateare good examples. ATP donates the phosphate moiety in ester formation. lClycoside bond formation (see below) and mutarotation(discussedalready) may also be referred to, as these are also the characteristic propertiesof monosaccharides.l GLYCOSIDES Glycosidesare formed when the hemiacetal or hemiketal hydroxyl group (of anomeric carbon)of a carbohydratereactswith a hydroxyl group of another carbohydrate or a non- carbohydrate (e.g. methyl alcohol, phenol, glycerol). The bond so formed is known as glycosidic bond and the non-carbohydrate moiety (when present)is referredto as aglycone. The monosaccharidesare held together by glycosidic bonds to result in di-, oligo- or polysaccharides(seelaterfor structures). H-C=O I _ + HrN-NH-CuHu H-C-OH R Glucose Phenylhydrazine H-C:N-NH-CoHs I H-C-OH I R Glucohydrazone l7-H2N-NH-C6H' I H-C:N-NH-CoHs I C:N-NH-CoHs I R Glucosazone Fig.2.10: A summatyof osazonefomation H-C-OH C----r tlll H-C-OH Conc.HeSoo H-Q L I rH I U H-C-OH '1 H-C I CHrou 3H2o H-d---l D-Ribose Furfural Fig.2.9 : Dehydration of monosaccharides with concentrated H "SO o. Dehydration When treatedwith concentratedsulfuricacid, monosaccharidesundergodehydrationwith an eliminationof 3 water molecules.Thus hexoses give hydroxymethylfurfuralwhile pentosesgive furfural on dehydration (Fi9.2.9).Thesefurfurals can condense with phenolic compounds (a-naphthol)to form coloured products.This is the chemical basisof the popular Molisch test. In case of oligo- and polysaccharides,they are firsthydrolysedto monosaccharidesby acid,and this is followed by dehydration. Osazone formation Phenylhydrazinein acetic acid, when boiled with reducing sugars, forms osazones in a reactionsummarizedin Fig,2,10. As is evident from the reaction, the first two carbons (Cr and C2) are involved in osazone formation. The sugars that differ in their configuration on these two carbons give the same type of osazones,since the differenceis maskedby bindingwith phenylhydrazine.Thus glucose,fructoseand mannosegive the same type (needle-shaped)osazones. Reducingdisaccharidesalso give osazones- maltose sunflower-shaped,and lactose powder- puff shaped. (RrcprcsentsCrto Crofglucose).
  • t8 BIOCHEMISTRY Naming of glycosidic bond : The nomenclatureof glycosidic bonds is based on the Iinkagesbetweenthe carbon atomsand the status of the anomeric carbon (o or p). For instance,lactose-which is formed by a bond between C1 of p-galactoseand Ca of glucose- is namedas 0(.1-+ 4) glycosidicbond. The other glycosidicbonds are describedin the structure of di- and polysaccharides. Physiologieally important glycosides 1. Glucovanillin (vanillin-D-glucoside)is a naturalsubstancethat impartsvanilla flavour. 2. Cardiac glycosides(steroidalglycosides): Digoxin and digitoxin contain the aglycone steroidand they stimulatemusclecontraction. 3. Streptomycin, an antibiotic used in the treatmentof tuberculosisis a glycoside. 4. Ouabain inhibits Na+- K+ ATPase and blocksthe activetransportof Na+. DERIVATIVESOF MONOSACCHARIDES Thereare severalderivativesof monosaccha- rides, some of which are physiologically important 1. Sugar acids : Oxidation of aldehyde or primaryalcoholgroupin monosaccharideresults in sugaracids.Cluconic acid is producedfrom glucose by oxidation of aldehyde (C1 group) whereasglucuronicacid is formedwhen primary alcoholgroup (C6)is oxidized. 2. Sugar alcohols (polyols) : They are producedby reductionof aldosesor ketoses.For instance,sorbitol is formed from glucose and mannitol from mannose. 3. Alditols : The monosaccharides, on reduction,yield polyhydroxyalcohols,known as alditols. Ribitol is a constituent of flavin coenzymes; glycerol and myo-inositol are componentsof lipids.Xylitol is a sweetenerused in sugarlessgumsand candies. 4. Amino sugars : When one or more hydroxyl groups of the monosaccharidesare replaced by amino groups, the products formed are amino sugarse.g. D-glucosamine, D-galactosamine.They are present as consti- tuentsof heteropolysaccharides. The amino groups of amino sugars are sometimes acetylated e.g. N-acetyl D-gluco- samrne. N-Acetylneuraminic acid (NANA) is a derivativeof N-acetylmannoseand pyruvicacid. It is an important constituentof glycoproteins and glycolipids.The term sialicacid is usedto include NANA and its other derivatives. Certain antibiotics contain amino sugars which may be involvedin the antibioticactivity e.g. erythromycin. 5. Deoxysugars: These are the sugarsthat contain one oxygen lessthan that presentin the parent molecule. The groups -CHOH and -CH2OH become-CH2 and -CH3 due to the absenceof oxygen.D-2-Deoxyriboseis the most important deoxysugarsince it is a structural constituentof DNA (in contrastto D-ribose in RNA). 6. L-Ascorbic acid (vitamin C) : This is a water-solublevitamin, the structureof which closelyresemblesthat of a monosaccharide. The structuresof selected monosaccharide derivativesare depictedin Fig.2.l1. Among the oligosaccharides,disaccharides are the most common (Fig.2,l2).As is evident from the name, a disaccharideconsistsof two monosaccharideunits(similaror dissimilar)held together by a glycosidic hond. They are crystalline,water-solubleand sweetto taste.The disaccharidesare of two types '1. Reducingdisaccharideswith free aldehyde or keto group e.g. maltose, lactose. 2. Non-reducingdisaccharideswith no free aldehyde or keto group e.g. sucrose,trehalose. Maltose Maltose is composed of two a-D-glucose unitsheldtogetherby cl (1 -+ 4) glycosidicbond. Thefreealdehydegrouppresenton C1of second glucoseanswersthe reducingreactions,besides
  • Ghapter & : CAFIBOHYDRATES 19 H-C:O I H-C-OH I HO-C-H I H-C-OH I H-C-OH I COOH D-Glucuronicacid OHH D-2-Deoxyribose cH2oH I H-C-OH I cH2oH Glycerol H NHz D-Glucosamine HOH myo-lnositol H3C-C--HN HOH N-Acetylneuraminicacid Fiq.2.11 : Structuresol monosaccharidederivatives(selectedexamples). the osazone formations (sunflower-shaped). Maltosecan be hydrolysedby dilute acid or the enzyme maltaseto liberate two moleculesof cr-D-glucose. ln isomaltose,the glucose units are held togetherby o (1 --+6) glycosidiclinkage. Cellobioseis another disaccharide,identical in structurewith maltose,exceptthat the former has p (1 -r 4) glycosidiclinkage.Cellobioseis formedduringthe hydrolysisof cellulose. Suoroee Sucrose(canesugar)isthe sugarof commerce, mostlyproducedby sugarcane and sugarbeets. Sucrose is made up of a-D-glucose and p- D-fructose.The two monosaccharidesare held togetherby a glycosidicbond (a1-+ B2),between Cj of c-glucose and C2 of B-fructose.The reducing groups of glucose and fructose are involvedin glycosidicbond, hencesucroseis a non-reducing sugar,and it cannot form osazones. Sucroseis the major carbohydrateproduced in photosynthesis. lt is transported into the storageorgansof plants (such as roots, tubers and seeds).Sucroseis the mostabundantamong the naturallyoccurring sugars.lt has distinct advantagesover other sugarsas a storageand transoortform. This is due to the fact that in sucrose,both the functional groups (aldehyde and keto)are held togetherand protectedfrom oxidativeattacks. Sucrose is an important source of dietary carbohydrate.lt is sweeter than most other commonsugars(exceptfructose)namelyglucose, lactoseand maltose.Sucroseis employed as a sweeteningagentin food industry.The intestinal enzyme-sucrase-hydrolysessucroseto glucose and fructosewhich are absorbed. F-aetsse Lactoseis more commonlv known as milk sugarsinceit is the disaccharidefound in milk. Lactoseis composed ol p-D-galactoseand B-D- glucoseheld togetherby 0 (1 -r a) glycosidic bond.The anomericcarbonof C1glucoseis free, hence lactoseexhibits reducing propertiesand formsosazones(powder-puffor hedgehogshape). Lactose of milk is the most important carbohydratein the nutritionof young mammals. It is hydrolysedby the intestinalenzyme lactase to glucoseand galactose. lnversion ef suerose Sucrose,as such is dextrorotatory(+66.5o). But, r,r,hen hydrolysed, sucrose becomes levorotatory(-28.2"). The processof change in optical rotation from dextrorotatory (+) to levorotatory(-) is referredto as inversion.The
  • BIOCHEMISTF|Y HOH Glucose Fructose Sucrose (a-D-glucosyl(1 --+2)p-D-fructose) Galactose Lactose (p-D-galactosyl(1 -+ a)p-D-glucose) Fig. 2.12 : Structures of disaccharides -maltose, sucrose and lactose. hydrolysed mixture of sucrose, containing gfucoseand fructose, is known as invert sugar. The processof inversionis explainedbelow. Hydrolysisof sucroseby the enzyme sucrase (invertasdor dilute acid liberatesone molecure each of glucoseand fructose.ft is postulatedthat sucrose (dextro) is first split into a-D- glucopyranose(+52.5") and p-D-fructofuranose, both being dextrorotatory. However, p-D- fructofuranoseis lessstableand immediatelygets converted to p-D-fructopyranose which is stronglylevorotatory(-92"). The overalleffectis that dextro sucrose (+66.5") on inversion is converted to levo form (28.2'. Polysaccharides(or simply glycans)consistof repeat units of monosaccharides or their derivatives,held togetherby glycosidicbonds. Theyareprimarilyconcernedwith two important functions-structural,and storageof energy. Polysaccharides are linear as well as branched polymers. This is in contrast to structureof proteinsand nucleicacidswhich are only linear polymers. The occurrence of branchesin polysaccharidesis due to the fact that glycosidic linkagescan be formed at any one of the hydroxylBroupsof a monosaccharide. Polysaccharidesare of two types 1. Homopolysaccharideswhich on hydrolysis yield only a singletype of monosaccharide.They are named based on the nature of the monosaccharideunit. Thus,glucans arepolymers of glucose whereas fructosans are polymers of fructose. 2. Heteropofysaccharideson hydrolysisyield a mixture of a few monosaccharidesor their derivatives. $tarch Starch is the carbohydrate reserveof plants which is the most importantdietary sourcefor higheranimals,includingman. High contentof starchis found in cereals,roots,tubers,vegetables etc. Starch is a homopolymer composed of D-glucoseunits held by a-glycosidicbonds. lt is known as glucosan or glucan. Starch consists of two polysaccharide components-water soluble amylose (15-20o/ol and a water insoluble amylopectin (80-85%). Chemically, amylose is a long unbranched chain with 200-1,00OD-glucoseunitsheld by c (1 + 4) glycosidiclinkages.Amylopectin,on the other hand, is a branchedchain with a (1 --r 6t glycosidicbondsat the branchingpointsand c (1 -; 4) linkages everywhere else (Fig.2.13). Amylopectin molecule containing a few
  • ChapteF 2 : CARBOHYDFATES 21 D-Glucose D-Glucose Amylopectin o-Amylose +- (1-* 6) Branch MainchainLg 6nu vt t2 thousandglucoseunits looks like a branched tree (20-30 glucoseunits per branch). Starches are hydrolysed by amylase (pancreaticor salivary)to liberatedextrins,and finally maltoseand glucoseunits.Amylaseacts specificallyon a (1 -+ 4) glycosidicbonds. Dextrins Dextrins are the breakdown products of starchby the enzyme amylaseor dilute acids. Starch is sequentially hydrolysed through different dextrins and, finally, to maltose and glucose.The variousintermediates(identifiedby iodine colouration) are soluble starch (blue), amylodextrin (violet), erythrodextrin (red) and achrodextrin (no colour). Inulin fnulin is a polymerof fructosei.e., fructosan. It occursin dahlia bulbs,garlic,onion etc. lt is a low molecularweight (around5,000) poly- saccharideeasilysoluble in water. Inulin is not utilized by the body. lt is used for assessing kidney function through measurement of glomerular filtration rate (GFR). Glycogen Clycogen is the carbohydrate reserve in animals,henceoften referredro asanimal starch. It is present in high concentration in liver, followed by muscle,brainetc.Clycogenis also found in plantsthat do not possesschlorophyll (e.9.yeast,fungi). The structureof glycogenis similarto that of amylopectin with more number of branches. Glucoseis the repeatingunit in glycogenjoined togetherby u (1 + 4) glycosidicbonds,and a (1 + 6) glycosidic bonds at branching points (Fi9.2.1Q.The molecularweight (up to 1 x 108) and the numberof glucoseunits (up to 25,000) vary in glycogendependingon the sourcefrom which glycogenis obtained.
  • 22 BIOCHEMISTRY Fiq.2.14: Structureofglycogen(A)Generalstructure (B)Enlargedat a branchpoint. Cellulose Celluloseoccursexclusivelyin plantsand it is the most abundant organic substancein plant kingdom. lt is a predominantconstituentof plant cell wall. Celluloseis totally absent in animal body. Cellulose is composed of p-D-glucose units linked by 9 0 -+ 4) glycosidic bonds(Fi9.2.1fl. Cellulosecannot be digestedby mammals- includingman-due to lack of the enzymethat cleavesB-glycosidicbonds(a amylasebreakscr bondsonly). Certainruminantsand herbivorous animalscontainmicroorganismsin thegutwhich produce enzymesthat can cleave p-glycosidic bonds. Hydrolysis of cellulose yields a disaccharide cellobiose, followed by P-D- glucose. Cellulose, though not digested, has great importancein human nutrition. lt is a major constituentol fiber, the non-digestablecarbo- hydrate.The functions of dietary fiber include decreasing the absorption of glucose and cholesterolfrom the intestine,besidesincreasing the bulk of feces. (For details,Chapter 23) Ghitin Chitin is composed of N-acetyl D- glucosamineunits held togetherby F (1 -+ a) glycosidicbonds.lt isa structuralpolysaccharide found in the exoskeletonof some invertebrates e.g. insects,crustaceans. When the polysaccharidesare composedof differenttypesof sugarsor their derivatives,they are referred to as heteropolvsaccharidesor heteroglycans. MUCOPOLYSACCHARIDES Mucopolysaccharidesare heteroglycansmade up of repeatingunitsof sugarderivatives,namely amino sugarsand uronic acids.Theseare more commonly known as glycosaminoglycans (GAG).Acetylatedamino groups,besidessulfate and carboxyl groups are generally present in CAC structure.The presenceof sulfate and carboxyl groups contributesto acidity of the molecules, making them acid mucopoly,- saccharides. Someof the mucopolysaccharidesare found in combination with proteins to forrn mucoproteins or mucoids or proteoglycans (Fig.2.l6l.Mucoproteinsmay containup to 95o, carbohydrate and 5o/"protein. S-D-Glucose T N T Ot (B) 9H2OH uqt, CH2oH y'-O., , F--o. ,r4-Or - (+ i) - r+ ,L^_K. X^_-oJ, ,./'o-'- J - L-/ " --l Fig. 2.15 : Structureof cellulose(The repeat:r; -- ' may be several thousands).
  • CARBOHYDRATES 23 Fig. 2.16 : Diagrammaticrepresentationof a prateoglycan complex. Mucopolysaccharidesareessentialcomponents of tissue structure.The extracellularspacesof tissue (particularlyconnective tissue-cartilage, skin, blood vessels,tendons)consistof collagen and elastinfibersembeddedin a matrixor ground substance.Thegroundsubstanceis predominantly composedof CAC. The importantmucopolysaccharidesincluoe hyaluronicacid, chondroitin4-sulfate,heparin, dermatansulfateand keratansulfate(Fig.Z.'[1. j'i' ,ir:r:, | '.i. :,{,tiiiEl'l Hyaluronicacid is an importantGAC found in the groundsubstanceof synovialfluid of joints and vitreoushumor of eyes.it is also presentas a ground substancein connectivetissues,and forms a gel aroundthe ovum. Hyaluronicacid servesas a lubricantand shock absorbantin joints. BToMEDtCAt/ CLtft|ICALCO$CEpTS Hyaluronicacid rlr Glucose is the most important energy sourceol carbohgdratesto the mammals (except ruminants).The bulk of dietary carbohydrote(starch)is dlgestedond finally obsorbedas glucose into the body. Ea Dextrose (glucosein solution in dextrorotatory form) is frequently used in medical Rq'- CF practice. Fructoseis obundantly found in the semen which is utilized by the spermsfor energy. Seueral diseoses are associated with carbohydrate.se.g., diabetes mellitus, glycogen storage diseoses,galactosemia. trs Accumulation of sorbitol and dulcitol in the fissuesmoy cause certoin pathological conditionse.g. cotaract, nephropothy. t-s' Inulin, a polymer of t'ructose,is used fo ossessrenal function by meosuringglomerular filtration rate (GFR). ue The non-digestiblecarbohydratecellulose playsa signilicant role in human nutriticsn. These include decreasing the intestinal absorption ol glucose and cholesterol, qnd increasingbulk of feces to ouoid eonstipation. rt The mucopolysaccharidehyaluronic acid seruesas a lubricant and shock absorbantin ioints. The enzgmehgaluronidaseof semendegradesthe gel (contains hyaluronic acid)around the ouum. This qllows eft'ectiuepenetration of sperm into the ouum. The mucopolysaccharideheparin is an onticoagulant(preuentsblood clotting). The suruiual of Antarctic lish below -2"C is attributed to the antit'reeze glycoproteins. streptomycin is a glycosideemployed in the treatment oJ tuberculosis. !3:. [j- IF s: -;-- s' -sS't/:- -'-
  • 24 BIOCHEMISTFIY Hyaluronic acid is composed of alternate units of D-glucuronic acid and N-acetyl D-glucosamine.These two molecules form disaccharideunits held togetherby 0 (t -+ S) glycosidic bond (Fi9,2,15).Hyaluronic acid containsabout 250-25,000 disaccharideunits (heldby p 1 -+ 4 bonds)with a molecularweight uo to 4 million. Hyaluronidase is an enzyme that breaks (B1 -+ 4 linkages)hyaluronic acid and other CAC. This enzyme is present in high concentrationin testes,seminalfluid, and in certainsnakeand insectvenoms.Hyaluronidase of semen is assignedan important role in fertilization as this enzyme clears the gel (hyaluronicacid) around the ovum allowing a better penetration of sperm into the ovum. Hyaluronidaseof bacteriahelps their invasion into the animaltissues. Ghondroitin sulfates Chondroitin 4-sulfate (Greek: chondros- cartilage) is a major constituent of various mammalian tissues(bone, cartilage,tendons, heart,valves,skin,corneaetc.).Structurally,it is comparablewith hyaluronicacid. Chondroitin 4-sulfateconsistsof repeatingdisaccharideunits composedof D-glucuronicacid and N-acetyl D-galactosamine4-sulfate(Fig.2.lV. Chondroitin5-sulfateis alsopresentin many tissues.As evident from the name, the sulfate group is found on C6 insteadof Ca. Heparin Heparin is an anticoagulant(preventsblood clotting)thatoccursin blood,lung,liver,kidney, spleenetc. Heparin helps in the releaseof the enzyme lipoprotein lipase which helps in clearingthe turbidityof lipemic plasma. Heparin is composedof alternatingunits of N-sulfoD-glucosamine6-sulfateand glucuronate 2-sulfate(Fi9.2.17). Dermatan sulfate The name dermatansulfateis derived from the fact that this compoundmostlyoccursin the skin. lt is structurallyrelated to chondroitin D-Glucuronicacid N-Acetylglucosamine Hyaluronic acid H NH-CO-CH3 N-Acetylgalactosamine 4-sulfate Chondroitin 4-sulfate o- D-Glucuronate-2-sulfateN-Sulfoglucosamine 6-sulfate Heparin t -o'r H NH_CO_CH. N-Acetylgalactosamine 4-sulfate Dermatansulfate H NH_CO :- N-Acetylglucosamine 6-sulfate Keratansulfate Fiq.2.17 : Structuresof commonglycosaminogi',-;-: - D-Glucuronicacid H O-SO; -o-so3 H NH-SOa qH2oH o the disaccharidesas repeatingunits.
  • Ghapter 2 : CAFIBOHYDHATES 25 Glycosaminoglycan Composition Tissuedistribution Function(s) Hyaluronicacid D-Glucuronicacid, N-acetylglucosamine Connectivetissue,synovialfluid, vitroushumor Servesasalubricant.and shockabsorber.Promotes woundhealing Chondroitinsulfate D-Glucuronicacid, N-acetylgalactosamine 4-sulfate Cartilage,bone,skin,bloodvessel walls Helpstomaintainthestructure andshapesoftissues Heparin D-Glucuronate2-sulfate,Blood,lung,liver,kidney,spleen N-sulfoglucosamine 6-sulfate Actsasananticoagulant Dermatansulfate L-lduronicacid,N-acetyl- galactosamine4-sulfate Bloodvesselvalves,heartvalves, Maintainstheshapesoftissues skin Keratansulfate D-Galactose,N-acetyl- glucosamine6-sulfate Cartilage,cornea,connective tissues Keepscorneatransparent 4-sulfate.The only differenceis that there is an inversion in the configuration around C5 of D-glucuronic acid to form L-iduronic acid (Fi9.2.1V. Keratan sulfate It is a heterogeneousCAG with a variable sulfate content, besides small amounts of mannose, fructose, sialic acid etc. Keratan sulfateessentiallyconsistsof alternatingunitsof D-galactosamine and N-acetylglucosamine 6-sulfate. A summaryof the glycosaminoglycanswith regardto composition,distributionand functions is given in Table 2.3. Several proteins are covalently bound to carbohydrateswhich are referredto as glyco- proteins. The carbohydrate content of glycoproteinvariesfrom 1o/oto 90o/oby weight, Sometimes the term mucoprotein is used for glycoprotein with carbohydrateconcentration more than 4"/o. Clycoproteins are very widely distributedin the cells and perform variety of functions.Theseincludetheir role as enzymes, hormones,transportproteins,structuralproteins and receptors.A selectedlist of glycoproteins and their major functionsis given in Table2.4. The carbohydratesfound in glycoproteins include mannose, galactose, N-acetyl- glucosamine, N-acetylgalactosamine,xylose, L-fucoseand N-acetylneuraminicacid (NANA). NANA is an importantsialicacid (SeeFig.2,l1). Antifreeze glycoproteins : The Antarctic fish live below -2oC, a temperatureat which the Glycoprotein(s) Major function(s) Collagen Hydrolases,proteases, glycosidases Ceruloplasmin lmmunoglobulins Synovialglycoproteins Thyrotropin,erylhropoietin Bloodgroupsubstances Fibronectin,laminin Intrinsicfactor Fibrinogen Structure Enzymes Transport Defenseagainstinfection Lubrication Hormones Antigens Cell-cellrecognitionand adhesion Absorptionofvitamin8,, Bloodclotting
  • 26 ElIOCHEMISTF|Y blood would {reeze.lt is now known that ihese fish contain antifreezeglycogtrateinwhich lower the freezingpoint of waterand interferewith tne crystalformationof ice. Antifreezegiycoproteins consistof 50 repeatingunits of the tripeptide, alanine-alawine-threonine. Each threonine residue is bound to B-galactosyl(1 + 3) o( N-acetylgalactosamine. ri#i .f*iCA# '? r,.4F.!"r.Ii $:F"r1.*fi { "'.3 i4 t: * :il The blood group antigens (of erythrocyte membrane) contain carbohydratesas glyco- proteinsor glycolipids.N-,A.cetylgaiactosamine, galactose,fucose,sialic acid etc. are found in the blood group substances.The carbohydrate contentalso playsa determinantrole in blood Eroup!n8. X. Carbohydrs,tesare the polyhydroxyaldehydesor ketones,or campounds whichproduce them on hydrolysis.The term sugor is applied to carbohydratessoluble in water and stDeetto taste. Carbahgdratesqre the major dietary energy sources,besidestheir inualuementin cell structure and uariousother t'unctions. 2. Carbohydrqtesare broadly c/ossiJiedinta 3 groups-ffionasqccharides,oligosoccharides and ytoiysaccharides.The monosacchsridesare further diuided into dit't'erentcategories bqsedan the presenceaf t'wnctionalgroups {oldosesar ketoses)and the number of carbon atoms (trioses,tetroses,pentases,hexosesand heptcses). 3. Glyceraldehyde{triose) is the simplestcarbohydrateand is chosen as a reJerenceto write the cont'iguratian of all other rnonasaccharides(D- anc L- forms). It' two rnonosaccharidesdiffer in their structurearound o singlecarbonatom, they ore known as eplmers.Glucoseand galactoseare C4-epimers. 4. D'Glucose is the most predominant naturally occurring aldosdmonosaccharide. Giucoseexisfscs a and p anemerswith dit'Jerentopticalrotations. The interconuersion of a and B anomericforms with changein theopticalrotatianis knoun asmutsratation. 5. Manosaccharidespariicipate in seuercl recctions"Theseinclude oxidation, reduction. dehydration, asazone formetion etc. Formatian ol esters and glycosides by manosacchqridesis af specialsignificanceln biochemical reactions. 6. Among the oligosacchqrides,disoccharidesare the most common. Theseinclude the reducing disaccharidesnamely lactose(rnilk sugar)and maltase(malt sugar)and the non-reducingsucrose(canesugar). 7. Palysacclwridesare the poiymersot' monosaccharidesor their deriuatiues,held together by glycosidic bonds.Homopalysaccharidessre compasedot' a single manosaccharicle (e.g., starch,glycogen,cellulose, inulin). Heteropolysaccharidescontain a mixture af Jew monasaceharidesor thetr derluatiues(e.g., rnucapolysacaharides). 8. Slorch and glgcogensre the carbohydratereseruesot' plants and animalsrespectiuelg. Cellulose,exclusiuelyt'ound in plants, is the structural constituent.Inulin is utilized to ossesskidney tunction bg measuringglomerular t'iltration rate (GFR). 9. Mucopoiysaccharides(glycosominoglycans)are the essential companents o/ tlssue structure. Theyprouide the mstrix or grownd substanceof extracellular tissuespacesin whtchcollagenand elastinfibers are embedded.Hyaluranic ocid,chondroitin 4'sult'ote, heporin, are amang the important glycosaminaglgcdns. 70. Glycoproteins are a group of biochernically important compaunds with a uariable composition of carbohyd.rate(7-900/o),caualently bound to protein. Seueralenzyrnes, hormanes,structura! proteinsand cellular receptorsare in fact glycoproteins.
  • Ghapter 2 : CAFIBOHYDHATES I. Essayquestions 1. Define and classifycarbohydrateswith suitableexamples.Add a note on the functionsof carbohydrates. 2. Describethe structureand functionsof mucopolysaccharides. 3. Cive an accountof the structuralconfigurationof monosaccharides,with specialreferenceto glucose. 4. Discussthe structureand functionsof 3 biochemicallyimportantdisaccharides. 5. Definepolysaccharidesand describethe structureof 3 homopolysaccharides. Short notes (a)Epimers,(b)Mutarotation,(c)Osazoneformation,(d)Clycosidicbond,(e)Sugarderivatives,(fl Anomers,(g)Enediol,(h)Amino su8ars,(i) Inversionof sucrose,(j) Deoxysugars. Fill in the blanks 1. Namea non-reducingdisaccharide 2. The carbohydratethat is taken as a referencefor writing the configurationof others 3. lf two monosaccharidesdifferin configurationarounda singlecarbonatom,they are known as 27 II. III. 4. 5. 6. 7. B. 9. 10. The s and B cyclicformsof D-glucoseare referredto as The non-carbohydratemoietyfound in glycosidesis known as Cive an exampleof a glycosideantibiotic Theglycosidicbondsat the branchingpointsin the structureof starchare The polysaccharideemployedfor the assessmentof kidneyfunction The glycosaminoglycanthat servesas a lubricantand shockabsorbantof joints Namethe sialicacid,mostlyfound in the structureof glycoproteinsand glycolipids IV. Multiple choice questions 11. Riboseand deoxyribosedifferin structurearounda singlecarbon,namely (a)Cr (b)Cz (c)C: (d)Cq. 12. One of the followingis not an aldose (a)Clucose(b)Calactose(c) Mannose(d) Fructose. 13. The glycosaminoglycanthat servesas an anticoagulant (a) Heparin(b) Hyaluronicacid (c)Chondroitinsulfate(d) Dermatansulfate. 14. The followingpolysaccharideis composedof B-glycosidicbonds (a)Starch(b)Clycogen(c) Dextrin(d)Cellulose. 15. The carbonatomsinvolvedin the osazoneformation (a)'l and 2 (b) 2 and 3 (c) 3 and 4 (d) 5 and 6.
  • Lirpirdls fl ?"'-o- i R--c-o1H frCH2-H R3 The Jat speaks : "ffith uater, I say, 'Touch menot': T'otlte tongue,I am tasteful; IY'ithin limits, I am datiful; fn excess,I am dangerous!" I ipids (Creek: lipos-fat) are of Breat L importance to the body as the chief concentratedstorageform of energy, besides their role in cellularstructureand variousother biochemicalfunctions.As such. lioids are a heterogeneous group of compounds ano, therefore,it is rather difficult to define them preciselv. Lipidsmay be regarded as organic substances relatively insoluble in water, soluble in organic solvents (alcohol, ether etc.), actually or potentially related to fatty acidsand utilized by the living cells. Unlike the polysaccharides,proteins and nucleic acids,lipids are not polymers.Further, lipidsare mostlysmall molecules. Lipids are broadlyclassified(modifiedfrom Bloor) into simple, complex, derived and miscellaneouslipids,whicharefurthersubdivided into differentgroups 1. Simple lipids: Estersof fatty acids with alcohols.Theseare mainly of two types (a) Fatsand oils (triacylglycerols): Theseare estersof fatty acids with glycerol. The difference between fat and oil is only physical.Thus,oil is a liquid while fat is a solid at room temperature. (b) Waxes: Estersof fattyacids(usuallylong chain)with alcoholsotherthan glycerol. These alcohols may be aliphatic or alicyclic.Cetylalcoholis mostcommonly found in waxes. 2. Complex(or compound)lipids: Theseare estersof fatty acids with alcohols containing additional groups such as phosphate, nitrogenousbase, carbohydrate,protein etc They are furtherdividedas follows (a) Phospholipids:They containphosphor,c acid and frequentlya nitrogenousbase This is in addition to alcohol and fai:. acids. 28
  • Chapter 3 : LIPIDS 29 (i) Glycerophospholipids: Thesephospho- lipids containglycerolas the alcohol e.9.,lecithin,cephalin. (ii) Sphingophospholipids: Sphingosineis the alcohol in this group of phospho- lipidse.g.,sphingomyelin. (b) Glycolipids: Theselipids contain a fatty acid, carbohydrateand nitrogenousbase. The alcohol is sphingosine,hence they are also called as glycosphingolipids. Clycerol and phosphateare absente.g., cerebrosides,gangliosides. (c) Lipoproteins: Macromolecularcomplexes of lipids with proteins. (d) Other complexlipids: Sulfolipids,amino- lipidsand lipopolysaccharidesareamong the othercomplex lipids. 3. Derived lipids: Theseare the derivatives obtainedon the hydrolysisof group 1 and group 2lipids which possessthe characteristicsof lipids.Theseincludeglycerolandotheralcohols, fatty acids,mono- and diacylglycerols,lipid (fat) soluble vitamins, steroid hormones, hydro- carbonsand ketonebodies. 4. Miscellaneouslipids: These include a large number of compounds possessingthe characteristics of lipids €.g., carotenoids, squalene,hydrocarbonssuch as pentacosane(in bees wax), terpenesetc. NEUTRAT LIPIDS: The lipids which are unchargedare referredto asneutrallipids.These are mono-, di-, and triacylglycerols,cholesterol and cholesterylesters. Functions of lipids Lipids performseveralimportantfunctions 1. They are the concentratedfuel reserveof the body (triacylglycerols). 2. Lipids are the constituentsof membrane structure and regulate the membrane permeability(phospholipidsand cholesterol). 3. They serve as a source of fat soluble vitamins(4, D, E and K). 4. Lipidsare importantas cellularmetabolic regulators(steroidhormonesand prostaglandins). 5. Lipidsprotectthe internalorgans,serveas insulatingmaterialsand give shapeand smooth appearanceto the body. Fatty acids are carboxylic acids with hydrocarbonside chain. They are the simplest form of lipids. Occurrence Fattyacidsmainly occur in the esterifiedform as major constituentsof variouslipids.They are also present as free (unesterified)fatty acids. Fattyacidsof animalorgin are much simplerin structure in contrast to those of plant origin which oftencontaingroupssuch asepoxy,keto, hydroxy and cyclopentanerings. Even and odd carbon fatty acids Most of the fatty acids that occur in natural lipids are of even carbons(usually 14C-2OC). This is due to the fact that biosynthesisof fatty acidsmainly occurswith the sequentialaddition of 2 carbon units. Palmitic acid (l6C) and stearicacid (l$C) are the most common. Among the odd chain fatty acids, propionic acid (3C) and valericacid (5C)are well known. Saturated and unsaturated fatty acids Saturatedfatty acids do not contain double bonds,while unsaturatedfattyacidscontainone or more double bonds. Both saturated and unsaturatedfatty acids almost equally occur in the natural lipids. Fatty acids with one double bond are monounsaturated,and thosewith 2 or more double bonds are collectivelv known as polyunsaturated fafty acids (PIJFA). Nomenclature of fatty acids The namingof a fatty acid (systematicname) is basedon the hydrocarbonfrom which it is derived. The saturatedfatty acids end with a suffix -anoic (e.g., octanoic acid) while the unsaturatedfatty acids end with a suffix -enoic
  • 30 BIOCHEMISTF|Y (e.9., octadecanoic acid). In addition to systematicnames/ fatty acids have common nameswhich are more widely used (Iable J. l). Numbering of carbon atoms : lt startsfrom the carboxylcarbonwhich is takenas number1. The carbonsadjacentto this (carboxylC) are2, 3, 4 and so on or alternatelya, F, T and so on. The terminalcarbon containingmethyl group is known omega (or) carbon. Starting from the methylend, the carbonatomsin a fattyacid are numberedas omega 1, 2, 3 etc. The numbering of carbon atoms in two different ways is given below 7654321 cH3 - cH2 - cH2- cH2-cH2 - cH2 - COOH 01 a2 o)3 ()4 ol5 (t)6 Length of hydrocarbon cha:n of fatty acids Dependingon the length of carbon chains, fatty acids are categorizedinto 3 groups-short chain with less than 6 carbons; medium chain with 8 to 14 carbons and long cfiain with 16 to 24 carbons. Shorthand representation of latty aclds lnstead of writing the full structures, biochemists employ shorthand notations (by numbers)to representfatty acids. The general rule is that the total numberof carbonatomsare written first,followed by the nunrberof double bonds and finally the (firstcarbon) position of Common Name Systematicname Abbreviationx Structure l.Saturatedfattyaclds Aceticacid Propionicacid Butyricacid Valericacid Caproicacid Caprylicacid Capricacid Lauricacid Myristicacid Palmiticacid Stearicacid Arachidicacid Behenicacid Lignocericacid Ethanoicacid n-Propanoicacid n-Butanoicacid n-Pentanoicacid n-Hexanoicacid n-Octanoicacid n-Decanoicacid n-Dodecanoicacid n-Tetradecanoicacid n-Hexadecanoicacid n-Octadecanoicacid n-Eicosanoicacid n-Docosanoicacid n-Tetracosanoicacid CHsCO0H CHgCHzCOOH CHs(CHz)z0O0H CHo(CHz)gCOOH CHs(CHe)+COOH CHe(CHz)oCOOH CHs(CHz)eC0OH CHs(CHz)roCOOH CHs(CHzhzCOOH CHg(CHz)t+CO0H CHs(CHz)roC0OH CHg(CHz)reCOOH CHs(CHz)zo00OH CH3(CHz)zzCOOH 2:0 3:0 4:0 F.n 6:0 8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0 24:0 ll. Unsaturatedfattyacids Palmitoleicacid Oleicacid Linoleicacid** Linolenicacid*x Arachidonicacid cr1s9-Hexadecenoicacid cls-9-Octadecenoicacid cls,cls-9,12-Octadeca- dienoicacid Allce9,12,15-0cta- decatrienoicacid Allcls-5,8,11,14- 16:1;9 18:1;9 18:2;9,12 18:3;9,12,'15 20:4;5,8,11,14 CHg(CHz)sCH=CH(CHz)zCOOH CHs(CHz)zCH=CH(CHz)zCOOH CHg(CHz)+CH=CHCHzCH=CH(CHz)zCOOH CHoCHzCH=CHCHzCH=CHCHzCH =CH(CHz)zCO0H CHg(CHz)+CH=CHCHzCH=CHCHzCH Elc0:a!tr3e!o!1ci1___ __=9H9'tcl=_cl9F!)49oli * Totalnunberofcarbonatons,followedbythenumberotdoublebondsandthefirctcarbonposrtionotthedoublebond(s). ** Essentialfawacids.
  • Ghapten 3 : LIPIDS 31 double bonds, startingfrom the carboxyl end. Thus,saturatedfattyacid, palmiticacid iswritten as.l 6:0, oleic acid as 18:1;9, arachidonic acid as 20 : 4; 5, 8, 11, 14. There are other conventionsof representing the double bonds.Ae indicatesthat the double bond is between9 and 10 of the fatty acid. o 9 representsthe double bond position(9 and 10) from the <oend. Naturallyoccurringunsaturated fatty acids belongto ro 9, ol 6 and o 3 series. a 3 series Linolenicacid(18 : 3;9, 12, 15) a 6 series Linoleicacid ('l8 : 2; 9, 12) and arachidonic acid (20 : 4; 5, 8, 11, 14) ro9 series Oleicacid(18 : 1; 9) The biochemically important saturatedand unsaturated fatty acids are given in the Table 3.1. The fatty acidsthat cannotbe synthesizedby the body and, therefore, should be supplied in the diet are known asessentialfattyacids(EFA). Chemically, they are polyunsaturated fatty acids, namely linoleic acid (18 : 2; 9, 12) and Iinolenic acid (18 : 3; 9, 12, 15). Arachidonic acid (20 :4;5,8, 11,14) becomesessential,if its precursorlinoleic acid is not providedin the diet in sufficientamounts.The structuresof EFA are given in the Table3.1. Biochemical basis for essentiality: Linoleic acid and linolenic acid are essentialsince humans lack the enzymesthat can introduce double bonds beyond carbons9 to 10. Functionsof EFA: Essentialfatty acids are required for the membrane structure and function, transportof cholesterol,formation of lipoproteins,preventionof fatty liver etc. They are also needed for the synthesisof another important group of compounds, namely eicosanoids(Chapter 32. Deficiency of EFA: The deficiency of EFA results in phrynoderma or toad skin, characterizedby the presenceof hornyeruptions H..ar(CHz)zCOOH H'c'1cHr;rcu, Oleic acid (clsform) Fig. 3.1 : Cis-trans isomerism in unsaturated fattv acids. on the posteriorand lateralpartsof limbs,on the back and buttocks,lossof hair and poor wound healing. lsomerism in unsaturated fatiy aeids Unsaturated fatty acids exhibit geometric isomerismdependingon the orientationof the groupsaround the double bond axis. lf the atomsor acyl groupsare presenton the same side of the double bond, it is a cis configuration. On the other hand, if the groups occur on the opposite side, it is a trans configuration. Thus oleic acid is a cis isomer while elaidic acid is a transisomer,as depicted in Fig.3.1. Cis isomersare lessstablethan frans isomers. Most of the naturally occurring unsaturatedfatty acids exist as crs isomers. In the cis isomericform, there is a molecular binding at the double bond. Thus, oleic acid exists in an L-shapewhile elaidic acid is a straightchain.Increasein the numberof double bonds will cause more bends (kinks) and arachidonicacid with 4 doublebondswill have a U-shape.lt is believed that cis isomersof fatty acids with their characteristic bonds will compactlypack the membranestructure. Hydroxy fatty acids: Someof the fatty acids are hydroxylated.p-Hydroxybutyricacid, one of the ketonebodiesproducedin metabolism,is a simple example of hydroxy fatty acids. Cerebronic acid and recinoleic acid are long chain hydroxy fatty acids. Cyclic fatty acids: Fatty acids with cyclic structuresare ratherraree.g./ chaulmoogric acid found in chaulmoogra oil (used in leprosy treatment)containscyclopentenylring. Elaldicacid (fransform)
  • 32 BIOCHEMISTFIY U A CH2-O-C Fl, ltl R2-C-O-CH O ttl cH2-o-c-R3 Triacylglycerol o cH2-o-c -B t- HO_CH I cH20H 1-Monoacylglycerol o o Rz-C cH2-o-c-R, -o-cH I cH2oH 1,2-Diacylglycerol O CH,_OH ill R-C-O-CH I cH2oH 2-Monoacylglycerol Fig. 3.2 : General structures of acylglycerols (For palmitoyl R = CtsHati for stearoyl R = C.rzHssiFor linoleoyl R = qtHsi Eicosanoids:Thesecompoundsare relatedro eicosapolyenoicfatty acids and include prosta- glandins,prostacyclins,leukotrienesand throm- boxanes.Theyarediscussedtogether(Chapter32). Triacylglycerols (formerly triglycerides) are the estersof glycerol with fatty acids. The fats and oils thatarewidely distributedin both plants and animals are chemically triacylglycerols. They are insolublein water and non-polarin characterand commonly known as neutralfats. Fatsas stored fuel : Triacylglycerolsare the most abundantgroup of lipids that primarily function as fuel reservesof animals. The fat reserveof normal humans (men 2Oo/o,women 25% by weigh$ is sufficientto meet the body's caloric requirementsfor 2-3 months. Fats primarily occur in adipose tissue: Adipocytes of adipose tissue-predominantly found in the subcutaneouslayer and in the abdominalcavity-are specializedfor storageof triacylglycerols.The fat is storedin the form of globulesdispersedin the entirecytoplasm.And surprisingly,triacylglycerolsarenot the structural componentsof biological membranes. Structures of acylglycerols: Monoacyl- glycerols, diacylglycerolsand triacylglycerols, respectivelyconsistingof one, two and three moleculesof fatty acidsesterifiedto a molecule of glycerol,are known (Fi5.3.2).Among these, triacylglycerols are the most important biochemically. Simpletriacylglycerolscontainthe sametype of fattyacid residueat all the threecarbonse.g., tristearoylglycerolor tristearin. Mixed triacylglycerols are more common. They contain2 or 3 different typesof fattyacid residues.In general,fatty acid attachedto C1 is saturated,that attached to C2 is unsaturated while that on C3 can be either.Triacylglycerols are named according to placement of acyl radicalon glycerole.9.,'l ,3-palmitoyl2-linoleoyl glycerol. Triacylglycerols of plants, in general, have higher content of unsaturated fatty acids compared to that of animals. $tereospecific numbering of glycerol The structureof glycerolgivesan impression thatcarbons1 and 3 are identical.Thisis not true in a 3-dimensionalstructure.In orderto represent the carbonatomsof glycerolin an unambiguous manner, biochemists adopt a stereospecific numbering(sn)and prefixglycerolwith sn. 6n,on no-C'.-H 6tr,ot sn-GfcJrol
  • C*rapter'3: LIPIDS 33 It should be noted that C1 and C3 are different. Cells possess enzymes that can distinguish these two carbons. Thus glycerokinasephosphorylatessn-3(andnot sn-l) glycerolto give sn-glycerol3-phosphate. PROPERTIESOF TRIACYLGTYCEROLS A few importantpropertiesof triacylglycerols, which have biochemical relevance, are discussedbelow 1. Hydrolysis: Triacylglycerols undergo stepwiseenzymatichydrolysisto finally liberate free fatty acids and glycerol. The processof hydrolysis,catalysedby lipasesis importantfor digestionof fat in the gastrointestinaltract and fat mobilizationfrom the adiposetissues. 2. Saponification: The hydrolysisof triacyl- glycerolsby alkalito produceglyceroland soaps is known as saoonification. Triacylglycerol+ 3 NaOH ---------+ Clycerol+ 3 R-COONa(soaps) 3. Rancidity: Rancidityis the term used to represent the deterioration of fats and oils resultingin an unpleasanttaste.Fatscontaining unsaturatedfatty acids are more susceptibleto ranciditv. Rancidity occurs when fats and oils are exposed to air, moisture, light, bacteria etc. Hydrolytic rancidity occurs due to partial hydrolysis of triacylglycerols by bacterial enzymes.Oxidativerancidityis due to oxidation of unsaturatedfatty acids. This results in the formation of unpleasant products such as dicarboxylic acids, aldehydes, ketones etc. Rancid fats and oils are unsuitablefor human consumotion. Antioxidants : The substanceswhich can preventthe occurrenceof oxidativerancidityare known as antioxidants. Trace amounts of antioxidantssuch as tocopherols(vitamin E), hydroquinone,gallic acid and c,-naphtholare addedto the commercialpreparationsof fatsand oilsto preventrancidity.Propylgallate,butylated hydroxyanisole(BHA) and butylated hydroxy- toluene(BHT)are the antioxidantsused in food preservation. a. tipid peroxidation in vivo: In the living cells, lipids undergo oxidation to produce peroxidesand free radicalswhich can damage the tissue.Thefreeradicalsarebelievedto cause inflammatory diseases, ageing, cancer/ atherosclerosisetc. lt is fortunatethat the cells possessantioxidantssuchasvitamin E,urateand superoxidedismutaseto prevent in vivo lipid peroxidation (Chapter 34). Tests to check purity of fats and oils Adulterationof fatsand oils is increasingday by day. Several tests are employed in the laboratoryto check the purity of fats and oils. Some of them are discussedhereunder lodine number: lt is defined as the grams (number) of iodine absorbedby 100 g of fat or oil. lodine number is usefulto know the relative unsaturationof fats,and is directly proportional to the content of unsaturatedfatty acids. Thus lower is the iodine number,lessis the degreeof unsaturation.The iodine numbersof common oils/fatsare given below. FaUoil lodine number Coconutoil Butter Palmoil Oliveoil Groundnutoil Cottonseedoil Sunfloweroil Linseedoil 7- 10 25- 28 4C- 55 80- 85 85- 100 100- 110 125- 135 175-200 Determinationof iodinenumberwill help to know the degreeof adulterationof a given oil. Saponificationnumber: lt is defined as the mg (number) of KOH required to hydrolyse (saponify)one gram of fat or oiL Saponification number is a measureof the averagemolecular sizeof the fattyacidspresent.Thevalueis higher for fats containing short chain fatty acids. The saponificationnumbersof a few fatsand oils are given below Humanfat : 195-200 Butter :230-240 Coconutoil : 250-260
  • 34 ElIOCHEMISTRY Reichert-Meissl(RM) number: lt is definedas the number of ml 0.1 N KOH required to completelyneutralizethe soluble volatile fatty acidsdistilledfrom 5 g fat. RM number is useful in testingthe purity of buttersince it containsa goodconcentrationof volatilefattyacids(butyric acid, caproicacid and caprylicacid).This is in contrastto other fats and oils which have a negligibleamount of volatile fatty acids. Butter hasa RM numberin the range25-30,while it is lessthan I for mostotheredibleoils. Thusany adulteration of hutter can be easily tested by this sensitiveRM number. Acid number : lt is definedas the numberof mg of KOH requiredto completely neutralize freefatty acidspresentin one gramfat or oil. In normalcircumstances,refinedoils shouldbe free from any free fatty acids. Oils, on decomoosition-due to chemical or bacterial contamination-yield freefatty acids.Therefore, oils with increasedacid number are unsafefor humanconsumption. These are complex or compound lipids containingphosphoricacid,in additionto fatty acids,nitrogenousbaseand alcohol(Fig.3.3). There are two classesof phospholipids 1. Clycerophospholipids(or phosphoglyce- rides)that contain glycerolas the alcohol. 2. Sphingophospholipids(or sphingomyelins) that containsphingosineas the alcohol. 1.i t ".t .;:i r,. : . ,,,.i., i-l, Clycerophospholipidsare the major lipids thatoccur in biologicalmembranes.Theyconsist of glycerol 3-phosphateesterifiedat its C1 and C2 with fatty acids. Usually, C1 contains a saturated fatty acid while C2 contains an unsaturatedfatty acid. 1. Phosphatidicacid : This is the simplest phospholipid. lt does not occur in good concentration in the tissues. Basically, phosphatidicacid is an intermediatein the synthesisof triacylglycerolsand phospholipids. The other glycerophospholipidscontaining differentnitrogenousbasesor other groupsmay be regardedas the derivativesof phosphatidic acid. 2. Lecithins (phosphatidylcholine)zTheseare the mostabundantgroupof phospholipidsin the cell membranes.Chemically,lecithin (Creek : lecithos-egg yolk) is a phosphatidicacid with choline as the base. Phosphatidylcholines represent the storage form of hody's choline. * BtoMEDtCAL/ CLtNtCAt CONCEpTS os Lipids are important to the body as constituentsof membranes,sourceol fat soluble (A, D, E and K) uitaminsqnd metabolic regulators(steroid hormonesand prostaglandlns), e Triacylglycerols (fots) primarily stored in the adipose tissue ore concentrated t'uel reseruesof the body. Fatst'ound in the subcutoneoustissueand around certaln orgons serueos thermal insulators, se The unsaturatedfatty acids-linoleicand linolenic acid-<re essentiolto humans, the deficiencyof which cousesphrynodermo or toad skin. s The cyclicfatty acid, namelychoulmoogricocid,isemployedin the treatmentof leprosy. og Fqts and oils on exposureto ah; moisture, bacteriaetc. undergo rancidity (deterioration). Thts can be preuented by the addition ol certain antioxidants (uitamin E, hgdroquinone, gallic acid). w In food preseruation,antioxidants-namely propyl gallote, butylated hydroxyanisole and butylated hydroxytoluene--arecommonly used.
  • Chapter 3 : LIPIDS 35 oll g cH2-o-c-R1 ill RI-C-O-CH .:1 -l CH2-i-'-r'- i't (1)Phosphatldicacid ,11 ill i tz)Leclthln(phosphatidylcholine) ,E otl I CH2-O-C-R1 ill R2-C-O-qH rf CH2-C- --l-CH2-CH2-NH2 C- Ethanolamine (3)Cephalln(phosphatidylethanolamine) o tl ? cH2-o-c-Rl R2-c-o-?H {l CH2-r-:- = C-CHz-CH-COO- o .),f,l(5)Phosphatldylserlne myalnositol (4)Phosphatidyllnosltol A QH2-O-CF{=CH-Rlltl R2-C-O-CH .:1 CH2-t', -i' i----CHz-CH2-NH2 ,t__ C- Ethanolamine (6)Plasmalogen(phosphatidalelhanolamine) r, n-cH2 ? tr. ? Hc-o-c-R3 R4-C-O-CH2 (7)Cardlollpin(diphosphatidylglycerol) ? cH2-o-c-R1cH2-, R2-C-O-CH I H?-OH ^ CH2-.i ,: r-.'-CHe + l- ehospnatioytgty."ro,I lCeramid" _ (/t'soninoosrne$)> CH3-(CH2)12-CH:CH-CH-?H-NH-C-R ' , *.CHg r_-CHz-CHz-Nf9,Tt Choline 'n3 (8)Sphlngomyelln Fig. 3.3 : Sttucturesof phospholipids.
  • 36 BIOCHEMISTF|Y (a) Dipalmitoyl lecithin is an important phosphatidylcholinefoundin lungs,lt isa surface active agent and prevents the adherence of inner surface of the lungsdue to surfacetension.Respiratory distresssyndromein infantsis a disorder characterizedby the absenceof dipalmitoyl lecithin. (b) Lysolecithinis formed by removalof the fatty acid either at C, or C, of lecithin. 3. Cephafins (phosphatidylethanolamine): Ethanolamineis the nitrogenousbasepresentin cephalins,Thus,lecithinandcephalindifferwith regardto the base. 4. Phosphatidylinositol: The steroisomer myo-inositolis attachedto phosphatidicacid to givephosphatidylinositol(Pl).Thisisan important comDonentof cell membranes.The action of certain hormones(e.9.oxytocin, vasopressin)is mediatedthroughPl. 5. Phosphatidylserine:The amino acid serineis presentin this group of glycerophos- pholipids.Phosphatidylthreonineis alsofound in certaintissues. 6. Plasmalogens: When a fatty acid is attachedby an etherlinkageat C1 of glycerolin the glycerophospholipids, the resultant compound is plasmalogen. Phosphatidal- ethanolamineis the most imoortantwhich is similarin structureto phosphatidylethanolamine but for the ether linkage(in place of ester).An unsaturatedfatty acid occurs at C1. Choline, inositoland serinemay substituteethanolamine to give other plasmalogens. Z. Cardiolipin: lt is so named as it was first isolated from heart muscle. Structurally, a cardiolipin consists of two molecules of phosphatidicacid held by an additionalglycerol through phosphategroups. lt is an important componentof inner mitochondrialmembrane. Cardiolipin is the only phosphoglyceridethat possessesantigenic properties. Sphingomyelins Sphingosineis an amino alcohol presentin sphingomyelins(sphingophospholipids).They do notcontainglycerolat all. Sphingosineisattached by an amide linkageto a fatty acid to produce ceramide.The alcohol group of sphingosineis bound to phosphorylcholinein sphingomyelin structure(Fig.3.3).Sphingomyelinsare important constituentsof myelin and are found in good quantityin brain and nervoustissues. Action of phospholipases Phospholipasesare a group of enzymesthat hydrolysephospholipids.Thereare four distinct phospholipases(Ar, 42, C and D), eachone of them specificallyactson a particularbond. For details,refer lipid metabolism(Chapter l4). Functions of phospholipids Phospholipidsconstitutean importantgroup of compound lipids that performa wide variety of functions 1. In associationwith proteins,phospholipids form the structural components of membranes and regulatemembranepermeability. 2. Phospholipids (lecithin, cephalin and cardiolipin)in the mitochondriaare responsible for maintaining the conformation of electron transportchain components,and thus cellular respiration. 3. Phospholipidsparticipatein the absorption of fat from the intestine. 4. Phospholipids are essential for the synthesisof different lipoproteins,and thus participate in the transport of lipids. 5. Accumulationof fat in liver(fattyliver)can be preventedby phospholipids,hence they are regarded as lipotropic factors. 6. Arachidonicacid,an unsaturatedfattyacid liberated from phospholipids, serves as a precursorfor the synthesisof eicosanoids(prosta- glandins,prostacyclins,thromboxanesetc.). 7. Phospholipidsparticipatein the reverse cholesteroltransport and thus help in the removalof cholesterolfrom the body. 8. Phospholipidsact as surfactants(agenL. lowering surface tension). For instance dipalmitoylphosphatidylcholineis an importar: fung surfactant. Respiratory distresssyndrome ^ infantsis associatedwith insufficientproductio^ of this surfactant.
  • Chapter 3 r LIPIDS 37 Sphingosine loHloYIY] o-cH2 Fig. 3.4 : Structure ot galactosylceramide(R = H). Fot sulfagalactosylceramideR is a sulfatide (R = SOi-). 9. Cephalins,an importantgroupof phospho- lipids participatein blood clotting. 10. Phospholipids(phosphatidylinositol)are involvedin signaltransmissionacrossmembranes. ClycoIipids (glycosphingol ipids)are important constituentsof cell membrane and nervous tissues(particularlythe brain). Cerebrosidesare the simplestform of glycolipids.Theycontaina ceramide(sphingosineattachgdto a fatty acid) and one or more sugars.Galactocerebroside (galactosylceramide)and glucocerebrosideare the most importantglycolipids.The structureof galactosylceramideis givenin Fig3.a. lt contains the fatty acid cerebronic acid. Sulfagalactosylceramideis the sulfatide derived from galactosylceramide. Gangliosides: Theseare predominantlyfound in ganglionsand are the most complex form of glycosphingolipids.They are the derivativesof cerebrosidesand containone or moremolecules of N-acetylneuraminicacid (NANA), the most imoortantsialicacid. The structureof NANA is givenin carbohydratechemistry(ReferFig.2.l1. The most important gangliosidespresentin the brain are CM1, CM2, CD, and CT, (G representsgangliosidewhile M, D and T indicate rnono-, di- or tri- sialic acid residues, and the number denotes the carbohydrate sequence attached to the ceramide). The ganglioside,CM2 that accumulatesin Tay-Sachs diseaseis reoresentednext (outlinestructure). Ceramide IGlucose f Galactos tl N-Acetyl- N-Acetyl- galactosamine neuraminicacid Lipoproteinsare molecular complexes of lipids with proteins. They are the transport vehiclesfor lipids in the circulation.Thereare five types of lipoproteins,namely chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density Iipoproteins (HDL) and free fatty acid-albumin complexes. Their structure, separation, metabolismand diseasesare discussedtogether (Chapter l4). Steroids are the compounds containing a cyclic steroid nucleus (or ring) namely cyclopentanoperhydrophenanthrene (CPPP). lr consistsof a phenanthrenenucleus(ringsA, B and C) to which a cyclopentanering (D) is attached. The structure and numbering of CPPP are shown in Fi9.3.5.The steroidnucleusrepresents saturatedcarbons,unlessspecificallyshown as double bonds.The methyl side chains(19 and
  • 38 BIOCHEMISTF|Y Fig. 3.5 : Sttucturcs of steroids (A, B, C-Perhydro- phenanthrene; D-Cyclopentane). 18) attachedto carbons10 and 13 are shownas single bonds. At carbon 17, steroidsusually containa sidechain. There are severalsteroidsin the biological system.Theseinclude cholesterol,bile acids, vitamin D, sex hormones, adrenocortical hormones,sitosterols,cardiac glycosidesand alkaloids.lf the steroid,containsone or more hydroxyl groups it is commonly known as sterol (meanssolid alcohol). CI{OLESTEROL Chofesterol, exclusively found in animals, is the most abundantanimal sterol.lt is widely distributedin all cellsand is a major component of cell membranesand lipoproteins.Cholesterol (Creek: chole-bile)was first isolatedfrom bile. Cholesterolliterally means 'solid alcohol from bile.' Structure and occurrence The structure of cholesterol (C27Ha6O)is depictedin Fig.3.5.lt hasone hydroxylgroup at C3 and a double bond between C5 and C6. An 8 carbon aliphaticside chain is attachedto C17. Cholesterolcontains a total of 5 methyl Sroups. Due to the presence of an -OH group, cholesterolisweaklyamphiphilic.As a structural component of plasma membranes,cholesterol is an important determinant of membrane permeabilityr,properties. The occurrence of cholesterolis much higher in the membranesof sub-celIular organelles. Cholesterolis founi in associationwith fany acids to- form cholestervlesters(esterification occursat the OH group of C3). Properties and reactions: Cholesterol is an yellowish crystallinesolid. The crystals,under the microscope, show a notched (E) appearance.Cholesterolis insoluble in water and soluble in organic solvents such as chloroform,benzene,ether etc. Several reactions given by cholesterolare useful for its qualitative identification and quantitativeestimation.TheseincludeSalkowski's test, Liebermann-Burchardreaction and Zak's test. Functions of cholesterol: Cholesterolis a poor conductorof heat and electricity,since it has a high dielectric constant.lt is presentin abundance in nervous tissues.lt appearsthat cholesterolfunctionsas an insulatingcover for the transmissionof electricalimpulsesin the nervous tissue. Cholesterol performs several other biochemicalfunctionswhich include its role in membranestructureand function, in the synthesisof bile acids, hormones (sex and cortical) and vitamin D (for details, Refer Chapters 7 and l9). ERGOSTEROL Ergosteroloccursin plants.lt is alsofound as a structuralconstituentof membranesin yeast and fungi. Ergosterol(Fig.3.5)is an important precursorfor vitamin D. When exposedto light,
  • fTi Cfrraptee3 : LIPIDS 39 li I cH3(cHdn-coo- Hydrophobic Hydrophiic hydrocarbonchain carboxylgroup (tail) (head) (A) Fatty acid the ring B of ergosterolopensand it is converted to ergocalciferol, a compound containing vitamin D activity. Theothersterolspresentin plantcellsinclude stigmasterol and ftsitosterol. (C)Amphipathic lipid otl Hydrophobic HYdroPhilic tail head (B) Phospholipid As per definition,lipidsare insoluble(hydro- phobic) in water. This is primarilydue to the predominantpresenceof hydrocarbongroups. However, some of the lipids possesspolar or hydrophilicgroupswhich tend to be solublein water. Molecules which contain both hydrophobicand hydrophilicgroupsare known as amphipathic (Creek : amphi-both, pathos- passion). Examplesof amphipathiclipids: Among the lipids,fatty acids,phospholipids,sphingolipids, bile salts and cholesterol(to some extent)are amphipathicin nature. Phospholipidshavea hydrophilichead(phos- phategroup attachedto choline, ethanolamine, inositol etc.) and a long hydrophobictail. The generalstructureof an amphipathicIipid may be representedas a polar or hydrophilicheadwith a non-polaror hydrophobictail (Fig.3.6). Fattyacidscontaina hydrocarbonchain with a carboxyl (COO-) group at physiologicalpH. The carboxyl group is polar in nature with affinityto water (hydrophilic)while hydrocarbon chain of fatty acid is hydrophobic. Orientation amphipathic lipids: When the amphipathiclipids are mixed in water (aqueous phase), the polar groups (heads) orient themselvestowards aqueousphase while the non-polar (tails) orient towards the opposite directions.This leadsto the formation of mr'celles (Fi9.3.6).Micelle formation, facilitatedby bile salts is very important for lipid digestion and absorption (Chapter 8). Me*nhrane bilayers In caseof biologicalmembranes,a bilayerof lipidsis formedorientingthe polar headsto the Aqueous pnase OOOOC Aqueousphase ttttlltttl Nonpolarphase ooAqueousphase (E)Lipidbilayer Fig. 3.6 : Summary of amphipathic lipids in the formation of micelle and lipid bilayer. (D)Micelle
  • 40 BIOCHEMISTRY BIOMEDICAL/ CLIITIICALCOilCEPTS !ei- € g The phospholipid4ipalmitoyl lecithin-preuents the adherence of inner surface of the lungs, the absenceof which is ossociofed with respiratory disfress syndrome in infants. Cepholinsparticipate in blood clotting. The action of certain hormones is mediated through phosphatidylinositol. Phospholipidsare important for the synthesisand transport of lipoproteins ond reuerse tronsport ol cholesterol. Cholesterolis essentialfor the synfhesisol bile ocids,hormones(sexand cortical)and uitaminD. Lipoproteinsoccur in the membronestructure, besidesseruingos o meansol transport uehiclesfor lipids. Lipids are associatedwith certain disorders----obesityond atherosclerosis. outer aqueous phase on either side and the nonpolar tails into the interior (Fig.3.6.fhe formation of a lipid bilayer is the basis of membranestructure. Liposomes: They are producedwhen amphi- pathic lipids in aqueousmedium are subjected to sonification. They have intermittent aqueous phases in the lipid bilayer. Liposomes,in combinationwith tissuespecificantiBens,are used as carriersof drugsto tarBettissues. Emulsions: These are produced when non- polar lipids (e.g. triacylglycerols)are mixed with water. The particlesare largerin size and stabilized by emulsifying agents (usuallr amphipathic lipids), such as bile salts and phospholipids.
  • LIPIDS substancesrelatiuely inso,lublein water, soluble in organicactually or potentially related to totti-o"iJl'ord or" utilized 2 Lipids are crassifiedinto simpre(fats ,and.,oirs), c,omprex(phosphoripids,grgcolipids),deriued (fatty acids, steriodn"r_"n"rl and miscelloneous(carotenoids). 3' Fatty acidsare the maior constituentsof.uariousripids.saturatedand unsaturotedfattyacidsarmostequarsoccurin "tt"*i'1io11i, ri'['i":ur"rrirrated fattyacids(pr'FA) ii[i:"0"i:':;::i:i and tinoteni,o,id o," the.s'se,'tiaiioitv o,ia, thatneedto be 4' Triacylglycerols(simply fats)are the esters 7f glycerol with fatty acids.They are foundin adipose tissueand pr:imoriry ,*nrtior.o, ',u"li.r"rr"- oj oli^otr. seuerar tests(iodinenumber,RMnumber)areemprogedin tn" ioiorrJo"/iio"i"i'r"inepurityof fotsand oirs,5' Phosphotipidsare complex lipids cctntainin.g phosphoricacid. Glycerophospholipids ;;:I:,:Ji:'::;',:i;,:i:;,:ot andtheseinct-ude'r"'"it"'i-"intin, phosphatravrinoiitot, 6' sphingophospholipids(sp.hingornyelins)contain sphingosineas the alcohol in place oftJi:;fi,!:,ttvcerophosphoti;idsi.-ih-osphoripid;";,;;i;;';";:, constituentsof ptasma 7 cerebrosidesare the .simprestform of grycoripidswhich occur in the membranesofneruoustissue. Gangriosidesare predominontrg round in ti" gorstions. Theycontainone or more moteculesof N-acetylneuraminic.";,; ifi;:;r;:,)i." t 8. Steroidscontain th,eringcyclopentanoperhydrophenanthrene. Timportanceincrude. "nlutt"ii,""i,,tJ'ir,ar,r,rr-iir'n,";;;.'I::#r::t i!^i,"Jlfr,,iZ,,hormones-A steroid containing'or."or"*or. hydroxyr groupsis known as steror.9' cholesteraris the most abundqnt animarsterar.It containsone hydroxyrgroup (at cz),a double bond (c51) and an "igl,t co,rao.nside choin,ti"rn.a b cp. cholesterol-is 2."?;:::::'t:.":"T"#[::;:::;,:x:_i:ao,, ,,,",riiJ"ii,'"ioJ",,,,nesiso/bireacids, r0 The lipids that oor:::r both hvdrophobic(non -porar)and hydrophiric(porar) groupsareknown as amphipathic. rhese in,i"a" iori, o,,;;.;:;;;h;i;,:;:, sphinsoripidsand bite ';:;r:#::.''athic ripidson i^poioi constituentsin the biiaeersof the bioroqical
  • 42 BIOCHEMISTRY Essayquestions 1. Writean accountof classificationof lipidswith suitableexamples. 2. Describethe structureandfunctionsof phospholipids. 3. Discussthesaturatedandunsaturatedfattyacidsof biologicalimportance,alongwiththeirstructures. 4. Describethe structureof steroids.Add a noteon thefunctionsof cholesterol. 5. Discussthebiologicalimportanceof amphipathiclipids. Short notes (a)Structureof triacylglycerols,(b) Clycolipids,(c) Essentialfattyacids,(d) Cis-transisomerism, (e)Rancidity,(0 lodinenumber,(g)Phosphatidylinositol,(h) Sphingomyelins,(i) Steroidnucleus, (j)Micelles. I. II. III. Fill 1. 2. 3. 4. 5. 6. B. 9. 10. in the blanks Thelipidsthatfunctionasfuelreservein animals Theisomerismassociatedwith unsaturatedfatWacids Thecyclicfattyacidemployedin thetreatmentof leprosy Thelipidsthatarenotthe structuralcomponentsof biologicalmembranes Theprefixsnusedto representglycerol,snstandsfor Thenumberof mgof KOH requiredto hydrolyse1 g fator oil isknownas Thephospholipidthatpreventsthe adherenceof innersurfacesof lungs Thephospholipidthatproducessecondmessengersin hormonalaction NametheglycolipidscontainingN-acetylneuraminicacid Thesteroidscontaina cyclicringknownas IV. Multiple choice questions 11. Thenitrogenousbasepresentin lecithin (a)Choline(b)Ethanolamine(c)Inositol(d)Serine. 12. Thenumberof doublebondspresentin arachidonicacid (a)1 (b)2 (c)3 @)a. 13. Oneof thefollowingisanamphipathiclipid (a)Phospholipids(b)Fattyacid(c)Bilesalts(d)All of theabove. 14. Esterificationof cholesteroloccursat carbonposition (a)1 (b)2 (c)3 (d)4. 15. Namethetestemployedto checkthe purityof butterthroughtheestimationof volatilefattyacids (a)lodinenumber(b)Reichert-Meisslnumber(c)Saponificationnumber(d)Acidnumber.
  • rl trii, p rotuint are the most ahundant organic I molecules of the living system.They occur in every part of the cell and constituteabout 50'h of the cellular dry weight. Proteinsform the fundamentalbasisof structureand function of life. Origin of the wotrd 'protein' The term protein is derived from a Creek word proteiog meaning holding the first place. Berzelius(Swedishchemist)suggestedthe name proteinsto the group of organiccompoundsthat are utmost important to life. Mulder (Dutch chemist)in 1838 usedthe term proteinsfor the high molecularweight nitrogen-richand most abundant substancespresent in animals and olants. Functions of proteins Proteinsperforma greatvarietyof specialized and essentialfunctionsin the living cells.These functions may be broadly grouped as static (structural) and dynamic. Structuralfunctions: Certainproteinsperform brick and mortar roles and are primarily responsiblefor structureand strengthof body. Theseinclude collagen and elastinfound in bone matrix, vascularsystemand other organs and a-keratin presentin epidermaltissues. Dynamic functions : The dynamic functions of proteinsare more diversifiedin nature.These include proteins acting as enzymeq hormones, blood clotting factors, immunoglobulins, membrane receptors,storageproteins,besides their function in genetic control, muscle contraction,respirationetc. Proteinsperforming dynamicfunctionsare appropriatelyregardedas the working horsesof cell. Elermental cornposition clf Broteins Proteinsare predominantlyconstitutedbv five major elementsin the following proportion. Carbon Hydrogen Oxygen Nitrogen Sulfur 50 - 55% 6 - 7.3% 19 - 24% 13 - 19% 0 - 4o/" 43
  • 44 BIOCHEMISTFIY Besidesthe above, proteinsmay also contain otherelementssuchasP,Fe,Cu, l, Mg, Mn, Zn etc. The content of nitrogen, an essential componentof proteins,on an averageis l6%. Estimationof nitrogenin the laboratory(mostly by Kjeldahl'smethod is also usedto find out the amountof proteinin biologicalfluids and foods. Proteins are polymers of amano acids Proteinson completehydrolysis(with concen- trated HCI for several hours) yield L-cr-amino acids. This is a common propertyof all the proteins.Therefore,proteins are the polymers of Lq"-amino acids. STANDARD AMINO ACIDS As manyas300 aminoacidsoccur in nature- Of these, only 20-known as standard amino acids are repeatedly found in the structure of proteins, isolated from different forms of life- animal,plant and microbial.This is becauseof the universalnatureof the geneticcode available for the incorporationof only 20 amino acids when the proteinsare synthesizedin the cells. The processin turn is controlledby DNA, the geneticmaterialof the cell. Afterthe synthesisof proteins,some of the incorporatedamino acids undergomodificationsto form their derivatives. Amino acids are a group of organic compounds containing two functional groups- amino and carboxyl. The amino group (-NH2) is basicwhile the carboxylgroup (-COOH) is acidic in nature. General structure of amino acids The amino acidsaretermedascr-aminoacids, if both the carboxyl and amino groups are attachedto the samecarbon atom, as depicted below The a-carbon atom binds to a side chain representedby R which is differentfor each of the 20 aminoacidsfound in proteins.Theamino acids mostly exist in the ionized form in the biologicalsystem(shownabove). Optical isomers of amino acids lf a carbon atom is attachedto four different groups, it is asymmetricand thereforeexhibits optical isomerism.The amino acids (except glycine) possessfour distinct groups (R, H, COO-, NH;) held by c,-carbon.Thus all the amino acids(exceptglycinewhere R = H) have optical isomers. The structuresof L- and D-amino acids are written based on the configurationof L- and D-glyceraldehydeas shown in Fig.4.l. The proteinsare composedof L-c-amino acids. Glassification of amino acids There are different ways of classifyingthe amino acidsbasedon the structureand chemicat nature,nutritionalrequirement,metabolicfateetc. A. Amino acid classification based on the structure : A comprehensiveclassificationof amino acids is based on their structureand chemicalnature.Eachamino acid is assigneda 3 letter or 1 letter symbol. These symbolsare commonlyusedto representthe amino acidsin protein structure.The 20 amino acids found in proteinsare divided into sevendistinctgroups. ln Table 4.1, the differentgroups of amino acids,their symbolsand structuresaregiven.The salientfeaturesof differentgroupsare described next cHo I H-C-OH I cH2oH D-Glyceraldehyde R I H-C-NH2 I cooH D-Amino acid cHo I oH-c-H I cH2oH L-Glyceraldehyde R H2N-C-H I cooH L-Amino acid Fig.4.l : D- and L-formsof amino acid based on the structure ot glyceraldehyde. H I R-C-COOH I NHz Generalstructure H I R-C-COO- NHJ Existsasion
  • Chapter 4 : PFIOTEINSAND AMINOACIDS 45 Symbol 3 letters I letter Structure Specialgroup present l. Aminoacldswithaliphaticsidechalns 1. Glycine Gly 2. Alanine Ala 3. Valine Val 4. Leucine Leu 5. lsoleucine H-CH-COO- t+ NHi t'!r-.rz-QH-coo HgC *nl Branchedchain Branchedchain Branchedchain ll. Aminoacidscontainlnghydroxyl(-OH)groups 6. Serine Thr Ser 7. Threonine cH2-cH-coo- oH NHi H3C-CH-CH-COO- on rrFrt Seeunderaromatic Hydroryl Hydroryl HydroxylTyrosine Tyr Trble 4.1 contd. nerl page
  • 46 BIOCHEMISTF|Y Narne Symbol 3 letters I letter Structure Specialgroup present lll. Sulfurcontainingaminoacids 8. Cysteine Cys C 9. MethionineMet M cH2-cH-coo- SH NHi Sulfhydryl Thioether cH2-cH-coo- A rrtI Cystine I Disulfide cH2-cH-@o- l+ NHi cH2-cH2-cH-@o- b-cH. runt lV. Acidicaminoacidsandtheiramides 10.Asparticacid Asp 11.AsparagineAsn 12.Glutamicacid Glu 13.Glutamine Gln po -ooc-cH2-cH- r+ NHi @o H2N-C-CH2-CH-COO- 6 nnl YPct-ooc-cH2-cH2-?H-coo- NH; H2N-C- CHz-CH2- ?H-COO- o NHI p-Carboryl Amide yCarboryl Amide V, Basicaminoacids 14.Lysine e6YP CH2-CH2-CH>-CHt l; NHi q -CH-@O- e-Amino l+ NHi Lys 15.Arginine Arg NH- CH2- CH2- CH2- CH-@O- ?:*t; NHt NHz -rq-cH+-coo- IINHi Guanidino lmidazole16. Histidine His HNN t.blo 4.1 contd. nort pag€
  • ehaptee r* r PFIOTEINSAND AMINO ACIDS 47 Name Symbol 3 letters 1 letter Structure Specialgroup present Vl. Aromaticaminoacids 17.PhenylalaninePhe cH2-9H-COO- Nxt Benzeneorphenyl Phenol lndole 18.Tyrosine Tyr 19.TryptophanTrp W /- '*:/""-[Xt"o" 4-'ir----------c H2-cH- coo- u_/ ''l*r H Pynolidine I gH Pro Vll. lminoacid 20. Proline (Note: Bgroupisshowninred) 1. Amino acids with aliphatic side chains : These are monoamino monocarboxylic acids. This group consistsof the most simple amino acids-glycine, alanine, valine, leucine and isoleucine.The last three amino acids (Leu, lle, Val) contain branched aliphatic side chains, hence thev are referred to as branched chain amino acids. 2. Hydroxyl group containing amino acids : Serine, threonine and tyrosine are hydroxylgroup containingamino acids. Tyrosine-being aromatic in nature-is usuallyconsideredunderaromaticamino acids. 3. Sulfur containingamino acids : Cysteine with sulfhydryl group and methionine with thioethergroup are the two amino acids incorporatedduring the course of protein synthesis. Cystine, another importantsulfurcontainingaminoacid,is formedby condensationof two molecules of cysteine. 4. Acidic amino acids and their amides : Aspartic acid and glutamic acids are dicarboxylic monoamino acids while asparagine and glutamine are their resoectiveamide derivatives.All these four amino acids possessdistinctcodons for their incorporationinto proteins. 5. Basicamino acids: Thethreeamino acids lysine, arginine (with guanidino group) and histidine (with imidazole ring) are dibasic monocarboxylicacids. They are highly basic in character. 6. Aromatic amino acids : Phenylalanine, tyrosineand tryptophan(with indole ring)
  • 48 BIOGHEMISTF|Y are aromaticamino acids. Besidesthese, histidine may also be consideredunder this category. 7. lmino acids: Prolinecontainingpyrrolidine ring is a unique amino acid. lt has an imino group (=NH), insteadof an amino group(-NH2) found in otheraminoacids. Therefore,proline is an a-imino acid. B. Classification of amino acids based on polarity : Amino acids are classified into 4 groups basedon their polarity.The polarity in turn reflectsthe functionalrole of amino acidsin protein structure. 1. Non-polar amino acids : These amino acidsare also referredto as hydrophobic (water hating). They have no charge on the 'R' group.The amino acidsincluded in this group are- alanine, leucine, isoleucine, valine, methionine, phenyl- alanine,tryptophanand proline. 2. Polar amino acids with no charge on 'R' group : Theseamino acids,as such,carry no chargeon the 'R'group.They however possess groups such as hydroxyl, sulfhydryl and amide and participatein hydrogen bonding of protein structure. The simple amino acid glycine (where R = H) is alsoconsideredin this category. The amino acids in this group are- glycine, serine, threonine, cysteine, glutamine,asparagineand tyrosine. 3. Polaramino acidswith positive'R' group : The three amino acids lysine, arginine and histidineare included in this group. 4. Polaramino acidswith negative'R'group : The dicarboxylic monoamino acids- aspartic acid and glutamic acid are consideredin this group. C. Nutritional classificationof amino acids : The twenty amino acids (Iable 4.1) arerequired for the synthesisof variety of proteins, besides other biological functions.However, all these20 amino acidsneednot be takenin the diet. Based on the nutritionalrequirements,amino acidsare grouped into two classes+ssential and non- essential. 1. Essentialor indispensableamino acids : The amino acids which cannot be synthesized hy the body and, therefore, need to be suppliedthrough the diet are called essentialamino acids. They are required for proper Browth and maintenanceof the individual. The ten amino acidslistedbelow are essentialfor humans(and also rats): Arginine, Valine, Histidine, lsoleucine, Leucine, Lysine, Methionine, Phenyla- lanine,Threonine,Tryptophan. lThe code A.U HILL,MP., T. T.(firstletter of each amino acid) may be memorized to recall essentialamino acids. Other useful codes are H. VITTAL, LMP; PH. VILLMA, TT, PW TIM HALL and MATTVILPhLy.I Thetwo amino acidsnamelyarginineand histidine can be synthesizedby adults and not by growingchildren,hencethese are considered as semi-essential amino acids (remember Ah, to recall). Thus, 8 amino acidsareabsolutelyessentialwhile 2 are semi-essential. 2. Non-essential or dispensabte amino acids : The body can synthesizeabout '10 amino acidsto meetthe biologicalneeds, hence they need not be consumed in the diet. Theseare-glycine, alanine,serine, cysteine, aspartate,asparagine, glutamate, glutamine,tyrosineand proline. D. Amino acid classification based on their metabolic fate : The carbon skeleton of amino acids can serve as a precursorfor the synthesis of glucose(glycogenic)or fat (ketogenic)or both. From metabolic view point, amino acids are divided into three groups (for details, Refer Chapter lA. 1. Glycogenic amino acids : These amino acids can serve as precursors for the formation of glucose or glycogen. e.g. alanine,aspartate,glycine,methionineetc. 2. Ketogenic amino acids : Fat can be synthesizedfrom these amino acids. Two amino acids leucine and lysine are exclusivelyketogenic.
  • Ghapter 4 : PFIOTE|NSAND AMTNOACTDS 49 3. Glycogenic and ketogenic amino acids : The four amino acidsisoleucine,phenyl- alanine, tryptophan, tyrosine are pre_ cursorsfor synthesisof glucoseas well as fat. Selenocysteine - the 2i st amino acid As already stated, 20 amino acids are commonly found in proteins.ln recentyears,a 21staminoacid namelyselenocysteinehasbeen added. lt is found at the active sitesof certain enzymes/proteins (selenoproteins). e.g. gluta_ thione peroxidase,glycine reductase,5,-deio- dinase,thioredoxinreductase.Selenocysteineis an unusual amino acid containingthe trace elementseleniumin placeof the sulfuratom of cysteine. z-!H-coo- NHd Cysteine Incorporation of selenocysteine into the proteinsduring translationis carriedout by the codon namely UCA. lt is interestingto notethat UCA is normally a stop codon that terminates protein biosynthesis.Another unique feature ts that selenocysteineis enzymaticallygenerated from serinedirectlyon the tRNA (selenocvsteine- IRNA), and then incorporatedinto proteins. Pyrrolysine-the 22nd amino acid? : In tne year 2002, some researchershavedescribedyet another amino acid namely pyrrolysineas the 22nd amino acid presentin protein. The stop codon UAG can code for pyrrolysine. Properties of amino acids The amino acids differ in their physico- chemicalpropertieswhich ultimatelydetermrne the characteristicsof proteins. A. Physical propefiies 1. Solubility: Most of the amino acids are usuallysolublein waterand insolublein organic solvents. 2. Melting points: Amino acids generally melt at highertemperatures,often above200.C. 3. Taste: Amino acids may be sweet (Cly, Ala, Val), tasteless(Leu) or bitter (Arg, lle). Monosodium glutamate (MSC; ajinomoto) is used as a flavoringagent in food industrv,and Chinesefoods to increasetaste and flavor. ln some individuals intolerant to MSC, Chinese restaurant syndrome (brief and reversible flu_ like symptoms)is observed. 4. Optical properties: All the amino acids exceptglycinepossessoptical isomersdue to the presence of asymmetric carbon atom. Some amino acids also have a second asymmetric carbon e.g. isoleucine,threonine.The structure of L- and D-amino acids in comparisonwith glyceraldehydehas been given (SeeFig.4.t). 5. Amino acidsas ampholytes: Amino acids contain both acidic (-COOH) and basic (-NH2) groups. They can donate a proton or accepta proton,henceamino acidsare regarded as ampholytes. Zwitterion or dipolar ion : The namezwitter is derivedfrom the Germanword which means hybrid. Zwitter ion (or dipolar ion) is a hybrid molecule containing positiveand negativeionic grouPs. The amino acidsrarelyexistin a neutralform with free carboxylic (-COOH) and free amino (-NH2) groups.In stronglyacidic pH (low pH), the amino acid is positively charged (cation) while in stronglyalkalinepH (high pH), it is negativelycharged(anion).Eachamino acid has a characteristicpH (e.g. leucine, pH 6.0) at which it carries both positive and negative chargesand existsas zwitterion (Fig.a.Z. IsoelectricpH (symbol pl) is defined as the pH at which a molecule existsas a zwitterion or dipolar ion and carriesno net charge.Thus, the moleculeis electricallyneutral. The pl value can be calculatedby takingthe averagepKavaluescorrespondingto the ionizable groups.For instance,leucine has two ionizabre groups/and its pl can be calculatedas follows. -cH-coo- rinJ Selenocysteine a L p1=4!9.9 =6.s z
  • 50 BIOCHEMISTFIY R-C-COO- I NHz Anion (hishpH) + H I R-C-COO- I NHi Zwitterion (isoelectricpH) Fig. 4.2 : Existence of an amino acid as cation, anion and zwitterion. Leucine exists as cation at pH below 6 and anion at pH above 6. At the isoelectricpH (pl = 6.0), leucineis found as zwitterion.Thus the pH of the medium determinesthe ionic natureof amino acids. Forthe calculationof pl of amino acidswith more than two ionizablegroups,the pKasfor all the groupshave to be taken into account. Titration of amino acids : The existenceof differentionic formsof amino acidscan be more easily understoodby the titration curves. The graphic representationof leucine titration is depictedin Fi9.4.3.At low pH, leucineexistsin a fully protonatedform ascation.As the titration proceedswith NaOH, leucine losesits protons and at isoelectric pH (pl), it becomes a zwitterion. Further titration results in the formationof anionic form of leucine. Some more details on isoelectricpH are discussed under the properties of proteins 1p.60). E, Chemica! properties The general reactions of mostly due to the presence groups namely carboxyl (-COOH) group and amino (-NH2) group. Reactionsdue to -COOH group 1. Amino acidsform salts(-COONa) with basesand esters(-COOR') with alcohors. 2. Decarboxylation:Amino acids undergo decarboxylationto producecorresponding amines. R-CH-COO ----+R-CH2 + CO2 NHa NHT This reactionassumessignificancein the livingcellsdue to the formationof many biologically important amines. These includehistamine,tyramineand y-amino butyricacid (CABA)from the amino acids histidine, tyrosine and glutamate, respectively. 3. Reaction with ammonia: The carboxyl group of dicarboxylicamino acids reacts with NH3 to form amide Aspartic acid + NH, ------;Asparagine Glutamic acid + NH. ------+Clutamine H I R-C-COOH I NHz Amino acid H I R-C-COOH I, NHi Cation (lowpH) H I 14 13 12 11 F-CH-COO- I NHz I I pH7 6 5 3 2 1 0 0.5 1.0 1.5 2.0 -+ Eouivalentsof NaOH-=+ amtno of two acids are functional Fig, 4.3 : Titrationcurueof an aminoacid-leucine (R = (CH),-CH-CH,-; PK,= Dissociationconstant for COOH; pl = lsoelectric pH; pK, = Dissociationconstantfor NHI).
  • -d*-*d,hGhapter 4 : PFIOTEINSAND AMINO ACIDS Reactionsdue to -NH2 group 4. The amino groups behave as basesand combine with acids (e.g. HCI) to form salts(-NHiCl-). 5. Reaction with ninhydrin: The cr-amino acids react with ninhydrin to form a purple, blue or pink colour complex (Ruhemann's purple). Amino acid + Ninhydrin---+ Ketoacid+ NHr+COz+Hydrindantin Hydrindantin+ NH: + Ninhydrin-----+ Ruhemann'spurple Ninhydrin reactionis effectivelyusedfor the quantitativedeterminationof amino acids and proteins.(Nofe : Proline and hydroxyprolinegive yellow colour with ninhydrin). 6. Colour reactionsof amino acids: Amino acidscan be identifiedby specificcolour reactions(See Table 4.3). 7. Transamination: Transferof an amino group from an amino acid to a keto acid to form a new amino acid is a very important reaction in amino acid metabolism(detailsgiven in Chapter 1fl. 8. Oxidativedeamination: The amino acids undergooxidativedeaminationto liberate free ammonia (Refer Chapter l5). ]{ON.STANDARD AMINO ACIDS Besides the 20 standard amino acids (described above) present in the protein structure,there are severalother amino acids which are biologicallyimportant.Theseinclude the amino acid derivativesfound in proteins, non-proteinamino acidsperformingspecialized functionsand the D-aminoacids. A. Amino acid derivatives in proteins : The 20 standardamino acids can be incoroorated into proteinsdue to the presenceof universal genetic code. Some of these amino acids undergo specific modificationafter the protein svnthesisoccurs. These derivativesof amino acidsareverfrp- ntforproteinstructureand functions. Selected examples are given hereunder. . Collagen-the most abundant protein in mammals-contains 4-hydroxyproline and 5-hydroxylysine. . Histones-the proteins found in association with DNA-contain many methylated, phosphorylatedor acetylatedamino acids. . y-Carboxyglutamic acid is found in certain plasmaproteinsinvolvedin blood clotting. . Cystine is formed by combination of two cysteines. Cystine is also considered as derivedamino acid. B. Non-protein amino acids : These amino acids,althoughneverfound in proteins,perform severalbiologically importantfunctions.They may be either d-or non-cr-amino acids. A selectedlistof theseamino acidsalongwith their functionsis given in Table4.2. C. D-Amino acids : The vast majority of amino acidsisolatedfrom animalsand olantsare of L-category.Certain D-amino acids are also found in the antibiotics (actinomycin-D, valinomycin, gramicidin-S). D-serine and D-aspartate are found in brain tissue. D- Glutamic acid and D-alanine are present in bacterialcell walls. Amino acids usefu! as drugs Therea certainnon-standardamino acidsthat are used as drugs. . D-Penicillamine (D-dimethylglycine), a metaboliteof penicillin,is employed in the chelationtherapyof Wilson'sdisease.This is possiblesinceD-penicillaminecan effectively chelatecopper. . N-Acetylcysteineis usedin cysticfibrosis,and chronic renal insufficiencv,as it can function as an antioxidant. . Gabapentin (y-aminobutyrate linked to cvclohexane)is used as an anticonvulsant. r'
  • 52 BIOCHEMISTRY Amino acids Function(s) L cr,-Aminoacids Ornithine I Citrulline I I Arginosuccinicacidl Thyroxine I I TriiodothyronineJ S-Adenosylmethionine Homocysteine Homoserine 3,4-Dihydroryphenylalanine(DOPA) Creatinine Ovothiol Azaserine Intermediatesinthebiosynthesisofurea. Thyroidhormonesderivedfromtyrosine. Methyldonorinbiologicalsystem. Intermediateinmethioninemetabolism.Ariskfactorforcoronaryheart diseases Intermediateinthreonine,aspartateandmethioninemetabolisms. Aneurotransmitter,servesasaprecursortormelaninpigment. Derivedlrommuscleandexcretedinurine Sulfurcontainingaminoacidfoundinfertilizedeggs,andactsasan antioxidant Anantibiotic ll. Non-s,-aminoacids p-Alanine p-Aminoisobutyricacid yAminobutyricacid(GABA) &Aminolevulinicacid(ALA) Taurine ComponentofvitaminpantothenicacidandcoenzymeA Endproductofpyrimidinemetabolism. Aneurotransmitterproducedfromglutamicacid Intermediateinthesynthesisofporphyrin(finallyheme) Foundinassociationwithbileacids. Proteinsarethe polymersof L-cr-aminoacids. The structureof proteinsis rathercomplexwhich can be divided into 4 levels of organization (Fig.4.4): 1. Primarystructure: The linearsequenceof amino acids forming the backboneof proteins (polypeptides). 2. Secondary structure: The spatial arrangement of protein by twisting of the polypeptidechain. 3. Tertiarystructure: The threedimensional structureof a functionalorotein. 4. Quaternary structure: Some of the proteins are composed of two or more polypeptidechains referredto as subunits.The spatialarrangementof thesesubunitsis known as quaternarystructure. lThe structural hierarchy of proteins is comparablewith the structureof a building.The amino acids may be consideredas the bricks, the wall as the primarystructure,the twistsin a wall as the secondarystructure,a full-fledged self-containedroom as the tertiary structure.A buildingwith similarand dissimilarroomswill be the quaternarystructurel. The term protein is generally used for a polypeptide containing more than 50 amino acids. ln recent years, however, some authors have been using'polypeptide'even if the numberof amino acidsis a few hundreds.They prefer to use protein to an assembly of polypeptidechainswith quaternarystructure.
  • Chapter 4 : PROTEINSAND AMINOACIDS 53 Primary structure Secondary structure Tertiary structure Quaternary structure Fig.4.4: Diagrammaticrepresentationofproteinstructure (Note: Thefoursubunitsof tuvotypesin quaternarystructure). PRIMARYSTRUCTUREOF PROTEIN Eachproteinhasa uniquesequenceof amino acids which is determined by the genes contained in DNA. The primary structureof a protein is largelyresponsiblefor its function.A vast majority of genetic diseasesare due to abnormalitiesin the amino acid sequencesof proteins i.e. changes associatedwith primary structureof protein. The amino acid compositionof a protein determinesits physicaland chemicalproperties. Peptide bond Theamino acidsare heldtogetherin a protein by covalent peptide bonds or linkages.These bonds are rather strong and serve as the cementing material between the individual amino acids (consideredas bricks). Formation of a peptide bond: When the amino group of an amino acid combineswith Ihe carboxyl group oI another amino acid, a peptide bond is formed (Fig.a.D. Note that a dipeptidewill have two amino acids and one peptide (not two) bond. Peptides containing more than 10 amino acids (decapeptide)are referredto as polypeptides. Characteristics of peptide bonds: The peptide bond is rigid and planar with partial double bond in character.lt generallyexistsin trans configuration. Both -C=O and -NH groups of peptide bonds are polar and are involved in hydrogenbond formation. Writing of peptidestructures: Conventionally, the peptidechainsarewrittenwith the freeamino end (N-terminalresidue)at the left,and the free carboxylend (C-terminalresidue)at the right.The aminoacid sequenceis readfrom N-terminalend to C-terminal end. Incidentally, the protein biosynthesisalsostartsfromthe N-terminalamino acid. H +l H3N-C-COO- 'l Rl Aminoacid1 +l + Fl"N-C-COO- R2 Aminoacid2 Hzo HH +l I H3N-C-€O-l'iH-C-COO- tl R1 R2 Dipeptide Fig.4.5 : Fomation of a peptidebond.
  • BIOCHEMISTRY54 Shorthand to read PePtides: The amino acids in a PePtideor Protein are representedby the 3-letteror one letter abbreviation. This is the chemical shorthandto write proteins. Naming of peptides: For naming peptides, the amino acid suffixes -ine (glycine),-an (tryptophan),-afe Glutamate)are changed to -Yl with the exception of C-terminal amino acid. Thus a tripeptide composed of an N- terminal glutamate,a cysteineand a C-terminal glycineis calledglutamyl-cysteinyl-glycine. ln the Fig.4.6,the namingand representation of a tripeptideare shown. Dimensions of a PePtide chain : The dimensions of a fullv extended polypeptide chain are depictedin Fig.4.7.The two adjacent cr-carbonatomsare placedat a distanceof 0.36 nm. The interatomicdistancesand bond angles are also shown in this figure. Deterrnination of primary stvueture The primary structurecomprisesthe identi- fication of constituentamino acids with regard to their quality, quantity and sequence in a proteinstructure.A pure sampleof a proteinor a polypeptideis essentialfor the determination of primary structurewhich involves3 stages: 1. Determinationof aminoacidcomposition. fruN--glrt"t"te--cysteine-glycine-Coo- Aminoacidsin a Peptide E _ C _G Glu CYs - GIY Glutamyl- cysteinYl- glYcine Onelettersymbols Threelettersymbols Peptidename Fig.4.6: lJseofsymbolsinrepresentinga peptide (Note:Atripeptidewrth3 aminoacidsandtwopeptidebondsis shown;Free-NHtr is ontheteftwhilefree-COt is ontheright)' 2. Degradation of protein or polypeptide into smallerfragments. 3. Determinationof the aminoacidsequence' 1. Determination of amino acid composition in a protein : The protein or polypeptide is completely hydrolysed to liberate the amino acids which are quantitativelyestimated'The hydrolysismay be carriedout either by acid or alkali treatment or by enzyme hydrolysis' Treatment with enzymes, however results in smallerpeptidesratherthan amino acids. Pronase is a mixture of non-specific proteolytic enzymes that causes complete hydrolysisof proteins. Separation and estimation of amino acids: The mixtureof amino acids liberatedby protein hydrolysis can be determined by chromato- graphic techniques. The reader must refer Chapter 41 tor the separationand quantitative determinationof amino acids' Knowledge on Fig.4.7 : Dimensionsof a futlyertended polypeptidechain' (The-distancebetween two adiacenta-carbonatoms is A'36 nm)'
  • Chapter 4 : PROTEINSAND AMINOACIDS JJ lOrN{' tFr - -/ -No,Sanger'sreagent i prot"intabetting i Hydrolysis i-+fr"" aminoacids Edman'greagent i Proteinlabelling i Hydrolysisi Hyorolysls ,$ Polypeptide (-N-terminalM) R I N-CH-COO- Phenylthlohydantoln(PTH)- amino acld Iv ldontlfledby chromatography primarystructureof proteinswill be incomplete withouta thoroughunderstandingof chromato- graphy. 2. Degradationof proteininto smallerfrag- ments: Proteinis a largemoleculewhich is sometimescomposedof individualpolypeptide chains.Separationof polypeptidesis essential beforedegradation. (a)liberation of polypeptides:Treatment with urea or guanidinehydrochloride disruptsthe non-covalentbonds and dissociatesthe proteininto polypeptide units.Forcleavingthe disulfidelinkages betweenthe polypeptideunits,treatment with performicacid is necessary. (b)Numberof polypeptides:Thenumberof polypeptidechainscan be identifiedby treatmentof proteinwith dansylchloride. ItspecificallybindswithN-terminalamino acidsto form dansylpolypeptideswhich on hydrolysisyield N-terminaldansyl aminoacid.Thenumberof dansvlamino acidsproducedisequalto the numberof polypeptidechainsin a protein. (c) Breakdown of polypeptides into fragments: Polypeptidesare degraded into smallerpeptidesby enzymaticor chemicalmethods. Enzymaticcleavage: Theproteolyticenzymes such as trypsin, chymotrypsin,pepsin and elastaseexhibit specificityin cleaving the peptide bonds (Refer Fig.8.V. Among these enzymes/trypsin is most commonlyused.lt hydrolysesthe peptidebondscontaininglysine or arginineon the carbonyl(-C:O) side of peptidelinkage. Chemical cleavage: Cyanogen bromide (CNBr)is commonlyusedto splitpolypeptides into smallerfragments.CNBrspecificallysplits peptidebonds,the carbonylside of which is contributedby the aminoacidmethionine. 3. Determinationof amino acid sequence: Thepolypeptidesor theirsmallerfragmentsare convenientlyutilizedfor the determinationof sequenceof aminoacids.Thisisdonein a step- wise mannerto finally build up the orderof aminoacidsin a protein.Certainreagentsare employedfor sequencedetermination(Fig,4,A. Dlnltrophenyl(DNP)- amlnoacld II + ldentifled by chromatography Ftg. 4.8 : Sanger'sreagent(llluoro 2,4-dinitrobenzene)and Edman'sreagent(Phenylisothiocyanate)in the determination of amino acid sequence of a protein (AA-Amino acid).
  • 55 ElIOCHEMISTRY Sanger'sreagent:Sangerused 1-fluoro2, 4-dinitrobenzene(FDNB)to determineinsulin structure. FDNB specifically binds with N-terminalaminoacidto form a dinitrophenyl (DNP)derivativeof peptide.Thison hydrolysis yieldsDNP-aminoacid (N-terminal)and free aminoacidsfromthe restof the peptidechain. DNP-aminoacidcanbe identifiedbv chromato- graphy. Sanger'sreagenthas limited use sincethe peptidechainis hydrolysedto aminoacids. Edman'sreagent: Phenyl isothiocyanateis the Edman'sreagent.lt reactswith the N- terminalaminoacidof peptideto forma phenyl thiocarbamylderivative.On treatmentwith mild acid,phenylthiohydantoin(PTH)-aminoacid,a cyclic compoundis liberated.This can be identifiedby chromatography(Fig.a.A. Edman'sreagenthas an advantagesincea peptidecanbe sequentiallydegradedliberating N-terminalaminoacidsoneafteranotherwhich canbe identified.Thisisdueto thefactthatthe peptideas a whole is not hydrolysedbut only releasesPTH-aminoacid. Sequenator:Thisisan automaticmachineto determinethe amino acid sequencein a polypeptide(with around100 residues).lt is basedon the principleof Edman'sdegradation (describedabove).Aminoacidsaredetermined sequentiallyfrom N-terminalend. The PTH- amino acid liberatedis identifiedby high- performanceliquid chromatography(HPLC). Sequenatortakesabout2 hoursto determine eachaminoacid. Overlapping peptides ln the determinationof primarystructureof protein,severalmethods(enzymaticor chemical) aresimultaneouslyemployed.Thisresultsin the formationof overlappingpeptides.Thisisdueto thespecificactionof differentagentson different sitesin the polypeptide.Overlappingpeptides arevery usefulin determiningthe aminoacid sequence. Reverse sequencing technique It is the geneticmaterial(chemicallyDNA) which ultimatelydeterminesthe sequenceof aminoacidsin a polypeptidechain.By analysing the nucleotidesequenceof DNA that codesfor protein,it is possibleto translatethe nucleotide sequence into amino acid sequence. This technique,however,failsto identifythe disulfide bondsand changesthatoccur in the amino acids afterthe protein is synthesized(post-translational modifications). SECONDARYSTRUCTUREOFPROTEIN The conformation of polypeptide chain by twisting or folding is referredto as secondary structure.The amino acids are locatedclose to each other in their sequence.Two types of secondary structures, a-helix and p-sheef, are mainly identified. lndian scientist Ramachandran made a significant contribution in understandingthe spatialarrangementof polypeptidechains. u-l{elix a-Helixisthemosfcommonspiralstructure of protein. lt has a rigid arrangementof polypeptidechain. a-Helical structurewas proposedby PaulingandCorey(1951)whichis regardedas one of the milestonesin the biochemistryresearch.The salientfeaturesof s-helix(Fig.a.9aregivenbelow 1. The a-helix is a tightlypackedcoiled structurewith aminoacidsidechainsextending outwardfrom the centralaxis. 2. The a-helix is stabilizedby extensive hydrogenbonding.ltisformedbetweenH atom attachedto peptideN, and O atomattachedto peptideC. Thehydrogenbondsareindividually weakbutcollectively,theyarestrongenoughto stabilizethe helix. 3. All thepeptidebonds,exceptthefirstand last in a polypeptidechain, participatein hydrogenbonding. 4. Eachturnof a-helixcontains3.5 amino acidsand travelsa distanceof 0.54 nm. The spacingof eachaminoacidis 0.15nm. 5. a-Helix is a stableconformationformed spontaneouslywith the lowestenergy.
  • Chapten 4 : PR0TEINSAND AMINO ACIDS 57 6. The righthandedo-helixis morestable thanlefthandedhelix(arighthandedhelixturns in thedirectionthatthefingersof righthandcurl whenitsthumbpointsin thedirectionthehelix rises). 7. Certainaminoacids(particularlyproline) disruptthea-helix.Largenumberof acidic(Asp, Clu) or basic(Lys,Arg,His)aminoacidsalso interferewith o-helixstructure. p-Pleated sheet Thisis the secondtypeof structure(hencep after a) proposedby Pauling and Corey. p-Pleatedsheets (or simply p-sheets)are composedof two or more segmentsof fully extendedpeptide chains (Fig,4,10).ln the p-sheets,the hydrogen bonds are formed between the neighbouring segments of polypeptidechain(s). Parallel and anti.parallel p.sheets The polypeptidechainsin the p-sheetsmay be arrangedeither in parallel (the same direction)or anti-parallel(oppositedirection). Thisis illustratedin Fig.4,l0. p-Pleatedsheet may be formed either by separatepolypeptidechains (H-bonds are interchain)or a singlepolypeptidechainfolding backon to itself(H-bondsareintrachain). (B) N-Terminal C-terminal (C) N-Terminal - C-terminal C-Terminal# N-terminal F19.4.10: StructureofB-pleatedsheet(A)Hydrogen bondsbetweenpolypeptidechains(B) Parallelp-sheet (C)AntiparallelB-sheet.(Note: RedctrctesinA (A) Flg.4.9: Diagrammaticrepresentationofsecondary structureofprotein-a righthandeda-helix H I (l-lndicate -C-R groupsofaminoacids; dottedblue linesare hydrogen bonds; Note that only a few hydrogen bonds shown for clarity). I representemlnoacidskeleton-C-R l.
  • 58 BIOCHEMISTRY Ft,.4.11 : Diagrammaticrepresentationofa protein containinga-helixandB-pleatedsheet(blue). Occurrenceof p-sheets:Many proteins containp-pleatedsheets.As such,the cx-helix and p-sheetare commonlyfound in the same protein structure(Fig.4.ll). In the globular proteins,p-sheetsform the corestructure. Othertypesof secondarystructures:Besides the cr-and p-structuresdescribedabove, the p-bends and nonrepetitive(less organised structures)secondarystructuresarealsofoundin proteins. TERTIARY STRUCTURE OF PROTEIN The three-dimensional arrangement of protein structure is referred to as tertiary structure.lt is a compact structurewith hydrophobicsidechainsheldinteriorwhilethe hydrophilicgroupsare on the surfaceof the protein molecule.This type of arrangement ensuresstabilitvof themolecule. Bonds of tertiary structure: Besidesthe hydrogenbonds,disulfidebonds(-S-S), ionic interactions (electrostatic bonds) and hydrophobicinteractionsalsocontributeto the tertiarystructureof proteins. Domains: The term domain is used to representthe basicunits of proteinstructure (tertiary)and function.A polypeptidewith 200 aminoacidsnormallyconsistsof two or more domains. OUATERNARYSTRUCTUREOF PROTEIN A greatmajorityof theproteinsarecomposed of single polypeptidechains. Some of the proteins, however, consist of two or more polypeptideswhich may be identical or "unrelated. Suchproteinsaretermedasoligomers andpossessquaternarystructure.Theindividual polypeptidechainsare known as monomers, protomersor subunits.A dimer consitsol two polypeptideswhilea tetramerhasfour. Bonds in quaternary structure: The monomericsubunitsare heldtogetherby non- convalent bonds namely hydrogen bonds, hydrophobicinteractionsand ionicbonds. Importanceof oligomericproteins:These proteinsplaya significantrolein the regulation of metabolismandcellularfunction. Examplesof oligomericproteins: Hemo- globin, aspartatetranscarbomylase,lactate dehydrogenase. Bonds responsible for protein structure Proteinstructureis stabilizedby two typesof bonds-covalentand non-covalent. 1. Covalentbonds: Thepeptideanddisulfide bondsarethestrongbondsin proteinstructure. The formation of peptide bond and its chracteristicshavebeendescribed. Disulfidebonds:A disulfidebond(-5-O is formedby the sulfhydrylgroups(-SH) of two cysteine residues, to produce cystine Gig.a.l2A).Thedisulfidebondsmaybe formed in a singlepolypeptidechain or between differentpolypeptides.Thesebondscontributeto the structuralconformationand stabilityof proteins. 2. Non-covalentbonds: There are,mainly, fourtypesof non-covalentbonds. (a)Hydrogenbonds: The hydrogenbonds areformedby sharingof hydrogenatoms between the nitrogen and carbonyl oxygen of different peptide bonds (Fig,4.12D.Eachhydrogenbondisweak but collectivelythey are strong.A large numberof hydrogenbondssignificantly contributeto the proteinstructure. (b)Hydrophobicbonds:Thenon-polarside chainsof neutralaminoacidstendto be
  • chapter 4 : PFIOTEINSAND AMINO ACIDS 59 (A) NH-CH-CO,,'V,^. gHz I S I, q/sdne (- i CHz NH-CH-CO/'.:,,,, r,.//^,,^_ C-CH - N ././././ ill ?R1 ! 'RaO /^,,,,^- N-;-8,.^./././ NH- CH- CO,'^./,'," HC-CHs I CH^ lsoleucine I' '7 H33 I Leuclne /,'v'r,'v,^NH- COl"r,^r,^r,'. NH-CH-CO,,'.,,a..,, I Aspanate t He " Lvslna ( Hzh- I ,4/'.,/- NH-CH- CO,/././ closelyassociatedwith each other in proteins(Fig.a.l2Q.As such,theseare not true bonds. The occurrenceof hydrophobic forces is observed in aqueous environment wherein the moleculesareforcedto staytogether. (c)Electrostaticbonds: Thesebonds are formed bv interactions between negativelychargedgroups(e.g.COO-)of acidic amino acids with positively charged groups (e.g. -NHj) of basic amino acids (Fi9.4.12D). (d) Van der Waals forces: These are the non-covalent associations between electrically neutral molecules.They are formed by the electrostatic interactions due to permanentor induceddipoles. Examples of protein structure Structureof humaninsulin: Insulinconsists of two polypeptidechains,A and B (Fig.a.lA, TheA chainhasglycineat the N-terminalend and asparagineat the C-terminalend. The B chain has phenylalanineand alanineat the N- andC-terminalends,respectively.Originally, insulinis synthesizedas a singlepolypeptide preproinsulin which undergoes proteolytic processingto giveproinsulinandfinally insulin. The structuralaspectsof hemoglobinand colfagenare respectivelygiven in Chaptersl0 and 22. Methods to determine protein structure For the determinationof secondaryand tertiaryproteinstructures,X-raycrystallography is most commonly used. Nuclear magnetic resonance(NMR)spectraof proteinsprovides structuraland functionalinformationon the atomsandgroupspresentin the proteins. (B) (c) (D) 21 ti SS ll1 30 Bchaln Ftq.4.13: Diagrammaticrepresentationof humaninsulin structure 19 Flg,4.12: Majorbondsinproteinstructure(A) Dbultide bond(B)Hydrogenbonds(C)Hydrophicbonds
  • 60 BIOGHEMISTRY Methods for the isolation and purification of proteins Severalmethodsare employedto isolateand purifyproteins.Initially,proteinsarefractionated by usingdifferentconcentrationsof ammonium sulfateor sodium sulfate.Proteinfractionation may also be carriedout by ultracentrifugation. Protein separationis achieved by utilizing electrophoresis,isoelectricfocussing,immuno- electrophoresis,ion-exchangechromatography, gel-filtration,high performanceliquid chromato- graphy(HPLC)etc.Thedetailsof thesetechniques are describedin Chapter4l. PROPERTIES OF PROTEINS 1. Solubility: Proteins form colloidal solutionsinsteadof true solutionsin water.This is due to huge size of protein molecules. 2. Molecular weight : The proteinsvary in their molecular weights, which, in turn, is dependent on the number of amino acid residues.Each amino acid on an average contributesto a molecularweight of about 110. Majority of proteinsholypeptides may be composedof 40 to 4,000 amino acids with a molecular weight ranging from 4,000 to 440,00O.A few proteinswith their molecular weightsare listedbelow : fnsulin-5,700;Myoglobin-1,7OO;Hemoglobin- 64,450;Serumalbumin-69,000. 3. Shape: There is a wide variationin the proteinshape.lt may be globular(insulin),oval (albumin)fibrousor elongated(fibrinogen). 4. lsoelectric pH : lsoelectricpH (pl) as a propertyof amino acidshasbeendescribed.The nature of the amino acids (particularlytheir ionizablegroups)determinesthe pl of a protein. The acidic amino acids (Asp, Clu) and basic amino acids(His,Lys,Arg)stronglyinfluencethe pl. At isoelectric pH, the proteins exist as zwitterions or dipolar ions. They are electrically neutral(do not migratein the electricfield)with minimum solubility, maximum precipitability and least buffering capacity. The isoelectric pH(pl) for some proteinsare given here Pepsin-'|.1;Casein-4.6;Human albumin-4.7; Urease-S.0;Hemoglobin-6.7; Lysozyme-l1.0. 5. Acidic and basic proteins: Proteins in which the ratio (e Lys+ e Ard/(e Clu + e Asp) it greaterthan 1 are referredto as basic proteins. For acidic proteins,the ratio is lessthan 1. 6. Precipitationof proteins: Proteinsexistin colloidal solution due to hydration of polar groups(-COO-, -NHt, -OH). Proteinscan be precipitatedby dehydrationor neutralizationof polar groups. Precipitationat pl : The proteinsin general are least soluble at isoelectric pH. Certain proteins(e.9.casein)geteasilyprecipitatedwhen the pH is adjusted to pl (4.6 lor casein). Formationof curd from milk is a marvellous example of slow precipitationof milk protein, caseinat pl. This occursdue to the lactic acid produced by fermentationof bacteria which lowersthe pH to the pl of casein. Precipitationby saltingout: The processof proteinprecipitationby the additionalof neutral saltssuch as ammonium sulfateor sodium sulfate is known as saltingout. This phenomenonis explainedon the basisof dehydration of protein moleculesby salts.Thiscausesincreasedprotein- protein interaction, resulting in molecular aggregationand precipitation. The amount of salt required for protein precipitationdependson the size (molecular weighg of the protein molecule.In general,the higheris the proteinmolecularweight,the lower is the saltrequiredfor precipitation.Thus,serum globulins are precipitated by half saturation with ammoniumsulfatewhile albumin is precipitated by full saturation. Salting out procedure is convenientlyusedfor separatingserumalbumins from globulins. The addition of small quantitiesof neutral salts increasesthe solubility of proteins.This process called as nlting rn is due to the diminishedprotein-proteininteractionat low salt concentration. Precipitation by saltsof heavymetals: Heavy metal ions like Pb2+, Hg2+, Fe2+,Zn2+t Cd2+ cause precipitation of proteins. These metals
  • Ghapter 4 : PFIOTEINSAND AMINO ACIDS 51 beingpositivelycharged,when addedto protein solution(negativelycharged)in alkalinemedium resultsin precipitateformation. Precipitationby anionic or alkaloid reagents: Proteinscan be precipitatedby trichloroacetic acid,sulphosalicylicacid,phosphotungsticacid, picric acid, tannic acid, phosphomolybdicacid etc. By the addition of these acids, the proteinsexistingas cations are precipitatedby the anionic form of acids to produce protein- sulphosalicylate, protein-tungstate, protein- picrateetc. Precipitation by organic solvents: Organic solvents such as alcohol are good protein precipitatingagents.They dehydratethe protein molecule by removingthe water envelopeand causeprecipitation. 7. Colour reactionsof proteins : The proteins give severalcolour reactionswhich are often usefulto identifythe natureof the amino acids presentin them. Biuretreaction: Biuretisa compoundformed by heatingurea to 180"C. biuret test is not clearlv known, lt is believed that the colour is due to the formation of a copper co-ordinated complex, as shown below. otl tl o The presenceof magnesiumand ammonium ions interfere in the biuret test. This can be overcomeby usingexcessalkali. The colour reactionsgiven by proteinsdue to the presenceof specificamino acidsare givenin Table 4,3. These reactions are often useful to know the Dresenceor absenceof the saidamino acidsin the given protein. DENATURATION The phenomenon of disorganization of native protein structure is known as denaturation. Denaturationresultsin the loss of secondary, tertiaryand quaternarystructureof proteins.This involvesa change in physical,chemical and biological propertiesof protein molecules. Reaction Specificgroup or amino acid 1, Biuretreaction Twopeptidelinkages 2. Ninhydrinreaction a-AminoacidsWhen biuret is treated with dilute copper sulfatein alkalinemedium, a purple colour is obtained.This is the basisof biuret test widely usedfor identificationof proteinsand peptides. Biuret test is answered by compounds containingtwo or more CO-NH groups i.e., peptide bonds. All proteins and peptides possessingat least two peptide linkages i.e., tripeptides(with 3 amino acids) give positive biurettest.Histidineis the only amino acid that answersbiurettest.The principleof biurettestis conveniently used to detect the presenceof proteinsin biologicalfluids.The mechanismof NHe t- C:O NH C=O NHz 3. Xanthoproteic reaction Benzeneringofaromatic aminoacids(Phe,Tyr,Trp) 4. Milllonsreaction Phenolicgroup(Tyr) 5. Hopkins-Colereaction Indolering(Trp) 6. Sakaguchireaction Guanidinogroup(Arg) 7, NitroprussidereactionSulfhydrylgroups(Cys) 8. Sulfurtest 9. Pauly'stest Sulfhydrylgroups(Cys) lmidazolering(His) Phenolicgroups(Tyr)
  • 62 BIOCHEMISTFIY Denaturation. Nativeprotein Fiq.4.14 : Denaturationof a protein. Agents of denaturation Physical agents: Heat, violent shaking, X-ravs,UV radiation. Chemical agents : Acids, alkalies, organic solvents(ether,alcohol), salts of heavy metals (Pb, Hg), urea,salicylate. Gharacteristics of denaturation 1. The native helical structureof protein is lost (Fig.4.l4). 2. The primary structureof a protein with peptide linkages remains intact i.e., peptide bonds are not hydrolysed. 3. The protein losesits biologicalactivity. 4. Denaturedprotein becomes insoluble in the solventin which it was originallysoluble. 5 The viscosity ol denatured protein (solution) increaseswhile its surface tension decreases. 6. Denaturationis associatedwith increasein ionizableand sulfhydrylgroupsof protein.This is due to lossof hydrogenand disulfidebonds. 7. Denaturedproteinis moreeasilydigested. This is due to increasedexposure of peptide bonds to enzymes. Cooking causes protein denaturation and, therefore, cooked food (protein)is more easilydigested. 8. Denaturationis usually irreversible.For instance,omelet can be preparedfrom an egg (protein-albumin)but the reversalis not possible. 9. Careful denaturationis sometimesrever- sible (known as renaturation). HemoSlobin undergoes denaturation in the presence of salicylate.By removalof salicylate,hemoglobin is renatured. 10. Denaturedproteincannotbe crystallized. Coagulation: The term 'coagulum' refersto a semi-solid viscous precipitate of protein. lrreversibledenaturationresultsin coagulation. Coagulation is optimum and requires lowest temperatureat isoelectric pH. Albumins and globulins (to a lesser extent) are coagulable proteins. Heat coagulation test is commonly usedto detect the presenceof albumin in urine. Flocculation: lt is the process of protein precipitationat isoelectricpH. The precipitateis referredto as flocculum. Casein(milk protein) can be easily precipitatedwhen adjusted to isoelectric pH (4.6 by dilute acetic acid. Flocculation is reversible.On application of
  • PROTEINSAND AMINOACIDS 63 heat, flocculum can be converted into an irreversiblemass,coagulum. CLASSIFICATION OF PROTEINS Proteinsare classifiedin severalways. Three majortypesof classifyingproteinsbasedon their function, chemical nature and solubility properties and nutritional importance are discussedhere. : i ti ! Basedon the functionsthey perform,proteins are classifiedinto the following groups (with examples) 1. Structural proteins: Keratin of hair and nails,collagenof bone. 2. Enzymesor catalyticproteins: Hexokinase, pepsrn. 3. Transportproteins: Hemoglobin,serum albumin. 4. Hormonal proteins: Insulin, growth normone. 5. Contractileproteins: Actin, myosin. 6. Storageproteins: Ovalbumin,glutelin. 7. Genetic proteins: Nucleoproteins. 8. Defenseproteins: Snakevenoms,lmmun- oglobulins. 9. Receptorproteinsfor hormones,viruses. This is a more comprehensiveand popular classificationof oroteins.lt is based on tne amino acid composition,structure,shapeand solubility properties. Proteins are broadly classifiedinto 3 major Broups 1. Simple proteins: They are composedof only amino acid residues. 2. Conjugatedproteins: Besidesthe amino acids, these proteins contain a non-protein moiety known as prosthetic group or conjugatinggroup. 3. Derivedproteins: Thesearethe denatured or degradedproductsof simple and conjugated oroterns. The above three classes are further subdividedinto differentgroups.The summary of proteinclassificationis given in the Table4.4. BIOMEDICAL/ CLINICALCONCEPTS Proteins are the most abundant organic molecules ol life. Theg perform static (structurol)and dynamic functions in the liuing cells. The dynomic t'unctionsof proteins are highly diuersiliedsuch os enzymes,hormones, clotting factors, immunoglobulins,storageproteins and membrane receptors. oe Half of the amino acids(about 70) that occur in proteins haue to be consumed by humans in the diet, hence they qre essentlal. A protein is soid to be complete (or lirst class)protein if oll the essentialamino acids are present in the requiredproportion by the human body e.g. egg olbumin. Cooking resultsin protein denaturotionexposingmorepeptide bondsfor easydigestion. Monosodium glutamate (MSG) ts used os a flauoring agent in loods to increasetaste and flauour.ln someindiuidualsintolerant to MSG,Chineserestaurantsyndrome(brief and reuersiblellu-like symptoms)is obserued.
  • 64 BIOCHEMISTF|Y Scleroproteins Albumins Globulins Glutelins Prolamines Histones Globins Protamines Gollagens Elastins Keratins Nucleoproteins Glycoproteins Mucoproteins Lipoproteins Phosphoproteins Chromoproteins Metalloproleins Coagulated prot€ins Proteans Metaproteins Proteoses Peptones Polypeptides Peptides l. Simpleproteins (a)Globularproteins:Thesearesphericalor oval in shape,solublein wateror other solventsanddigestible. (i) Albumins: Soluble dilutesaltsolutions by heat. e.g. serum albumin, ovalbumin(e8d,lactalbumin(milk). (ii) Globulins:Solublein neutraland dilute salt solutionse.g. serum globulins,vitelline(eggyolk). (iii) Glutelins:Solublein diluteacidsand alkaliesand mostlyfound in plants e.g.glutelin(wheat),oryzenin(rice). (iv) Prolamines:Solublein 7O1"alcohol e.g. gliadin(wheat),zein (maize). (v) Histones: Stronglybasic proteins, solublein wateranddiluteacidsbut insolublein diluteammoniumhydro- xidee.g.thymushistones,histonesof codfishsperm. (vi) Globins: Theseare generallyconsi- deredalongwith histones.However, globinsarenotbasicproteinsandare not precipitatedby NH/OH. (vii) Protamines:Theyarestronglybasic and resemblehistonesbut smaller in size and soluble in NH4OH. Protaminesare also found in associationwith nucleic acids e.g. sperm proteins. (b) Fibrousproteins : Theseare fiber like in shape,insolublein water and resistantto digestion,Albuminoids or scleroproteins constitutethe most predominantgroup of fibrous proteins. (i) Collagens are connective tissue proteins lacking tryptophan. Collagens,on boiling with water or dilute acids, yield gelatin which is solubleand digestible. (ii) Elastins:Theseproteinsare found in elastic tissuessuch as tendons and arteries. (iii) Keratins: These are present in exoskeletalstructurese.g. hair, nails, horns.Human hair keratincontainsas much as 14% cysteine. 2. Coniugated proteins (a) Nucleoproteins: Nucleic acid (DNA or RNA) is the prostheticBroupe.g. nucleo- histones,nucleoprotamines. (b) Glycoproteins: The prostheticgroup is carbohydrate,which is less than 4"/" of protein, The term mucoprotein is used if the carbohydratecontent is more than 4o/o. e.g.mucin (saliva),ovomucoid(eggwhite). tn and water and coagulated
  • Chapter 4 : PROTEINSAND AMINO ACIDS 65 (c) Lipoproteins: Protein found in combinationwith lipidsasthe prosthetic groupe.g.serumlipoproteins,membrane lipoproteins. (d)Phosphoproteins:Phosphoricacid is the prostheticgroup e.g. casein (milk), vitelline(eggyolk). (e)Chromoproteins:Theprostheticgroupis coloured in nature e.g. hemoglobins, cytochromes. (0 Metalloproteins:Theseproteinscontainmetal ionssuchas Fe,Co, Zn, Cu,Mg etc.,e.g. ceruloplasmin(Cu),carbonicanhydrase(Zn). 3. Derived proteins: The derivedproteins are of two types.The primaryderivedare the denaturedor coagulatedor first hydrolysed productsof proteins.Thesecondaryderivedare the degraded(due to breakdownof peptide bonds)productsof proteins. (a)Primaryderivedproteins (i) Coagulatedproteins: Theseare the denatured proteinsproducedby agentssuch as heat,acids,alkalies etc.e.g.cookedproteins,coagulated albumin(eggwhite). (ii) Proteans: These are the earliest productsof protein hydrolysisby enzymes,dilute acids,alkaliesetc. whichareinsolubleinwater.e.g.fibrin formed fromfibrinogen. (iii) N,lgf6plsteins: Theseare the second stageproductsof proteinhydrolysid obtainedby treatmentwith slightly strongeracidsand alkaliese.g.acid andalkalimetaproteins. (b)Secondaryderivedproteins: Thesearethe progressivehydrolyticproductsof protein hydrolysis.These include proteoses, peptones,polypeptidesandpeptides. G. Nutritional classification of proteins Thenutritivevalueof proteinsis determined by the compositionof essentialamino acids (describedalready).Fromthenutritionalpointof view, proteinsareclassifiedinto 3 categories. 1. Completeproteins: Theseproteinshave all thetenessentialaminoacidsin the required proportionby the humanbodyto promotegood growth.e.g.eggalbumin,milk casein. 2. Partiatlyincompleteproteins:Thesepro- teinsarepartiallylackingoneor moreessential aminoacidsand hencecan promotemoderate growth.e.g. wheatand rice proteins(limiting Lys,ThO. 3. Incomplete proteins: These proteins completelylack one or moreessentialamino acids.Hencetheydo not promotegrowthat all e.g.gelatin(lacksTrp),zein (lacksTrp,Lys). BIOTOGICALLY IMPORTANT PEPTIDES Severalpeptidesoccur in the living orga- nismsthat displaya wide spectrumof bio- logicalfunctions.Generally,theterm'peptide'is appliedwhenthe numberof constituentamino acidsis lessthan 10. Someexamplesof bio- logicallyactivepeptidesandtheirfunctionsare describedhere. 1. Glutathione: lt isa tripeptidecomposedof 3 aminoacids.Chemically,glutathioneis y- glutamyl-cysteinyl-glycine.lt iswidelydistributed in natureand existsin reducedor oxidized states. 2G-SH=+ G=S-S-G Reduced Oxidized Functions: In a steady state, the cells generallymaintaina ratio of about 100/1of CSH to G-S-S-C. The reversibleoxidation- reductionof glutathioneisimportantformanyof its biologicalfunctions. . Clutathioneservesasa coenzymefor certain enzymese.B.prostaglandinPCE,synthetase, glyoxylase. r lt preventsthe oxidation of sulfhydryl (-SH) groups of several proteins to disulfide(-S-S-1 groups.Thisisessentialfor the protein function, including that of enzymes.
  • 66 BIOCHEMISTFIY It is believedthat glutathionein association with glutathionereductaseparticipatesin the formationof correctdisulfidebondsin several proteins. Clutathione (reduced)performs specialized functionsin erythrocytes (i) lt maintainsRBCmembranestructureand integrity. (ii) lt protects hemoglobin from getting oxidized by agentssuch as HzOz. Clutathione is involved in the transoortof amino acids in the intestine and kidney tubules via y-glutamyl cycle or Meister cycle (Refer Chapter 8). Glutathioneis involved in the detoxication process. The toxic substances (organo- phosphates,nitro compounds)are converted to mercapturicacids. Toxic amountsof peroxidesand free radicals produced in the cells are scavanged by glutathioneperoxidase(a seleniumcontaining enzyme). 2 GSH+ tjrorl99l!!9!5G - s - s - G + 2 H2o 2. Thyrotropin releasing hormone (IRFI) : lt is a tripeptidesecretedby hypothalamus.TRH stlmulatespituitarygland to releasethyrotropic normone. 3. Oxytocin: lt is a hormone secretedby posteriorpituitarygland and contains9 amino acids(nonapeptide).Oxytocincausescontraction of uterus. 4. Vasopressin(antidiuretic hormone, ADI{) t ADH isalsoa nonapeptideproducedby posterior pituitarygland. lt stimulateskidneysto retain water and thus increasesthe blood pressure. 5. Angiotensins:AngiotensinI is a decapep- tide (10 amino acids) which is convertedto angiotensinll (8 amino acids).The later has more hypertensive effect. Angiotensinll also stimulates the release of aldosterone from adrenalgland. EIOMEtrICAL/ CLIIhIICALCONCEPTE lg 6 sa Collagen is the most abundant protein in mammols.lt is rich in hydroxyproline and hydroxylysine. Seuerolbiologicollyimportant peptidesare known in the liuing orgonism.Theseinclude glutathione t'or the maintenonceof RBC structure ond integrity; oxytocin that caases uterus contraction; uosopressin that stimulates retentlon ol water by kidneys; enkephalins that inhibit the senseof poin in the brain. Antibioticssuch os actinomycin,gramicidin, bocitracinand tyrocidin are peptide in nature. yCarboxyglutamic acid is an amino acid deriuatiuefound in certain plasma proteins inuoluedin blood clotting. Homocysteine hos been implicated os o risk loctor in the onset ol coronary heart diseoses. Seueral non-protein amino ocidsol biological importonce are known. These include ornithine, citrulline and arginosuccinicacid (intermediotesol urea synthesis),thyroxine and triiodothyronine (hormones),and ftalanine (of coenzymeA). The protein-free liltrote of blood, required tor biochemical inuestigotions(e.g. urea, sugar)can be obtained by usingprotein precipitating agentssuch os phosphotungstlc acid ond trichloroaceticacid. Heot coagulationtestis mosfcommonlyemployedto detectthepre.senceof albumin in urine.
  • PFOTEINSAND AMINOACIDS 67 6. Methionine enkephalin: lt is a penta- peptidefound in the brain and has opiate like function.lt inhibitsthe senseof a pain. 7. Bradykininand kallidin: They are nona- and decapeptides,respectively.Bothof them act as powerful vasodilators.They are produced from plasmaproteinsby snakevenom enzymes. 8. Peptide antibiotics: Antibiotics such as gramicidin,bacitracin,tyrocidin andactinomycin are peptidein nature. 9. Aspartame: lt is a dipeptide (aspartyl- phenylalanine methyl ester), produceo by a combination of aspartic acid and phenylalanine.Aspartameis about 200 times sweeter than sucrose, and is used as a low-calorie artificial sweetner in softdrink industry. 10.Gastrointestinal hormones: Castrin, secretin etc. are the gastrointestinalpeptides which serveas hormones. I. Protelnsare nltrogen containing, mostabunddnt organlcmscromoleculeswtdely distributed in animalsond plants.Theyperform structurol and dynamic lunctionsin the organisms. 2. Proteins are polymers composed ol L-a-amino acids. They are 20 in number and classifled into dtlt'erent groups based on their structure, chemical nature, nutritional requirementond metabolicfote. Selenocysteinehss beenrecentlyidentified as the 27st amlno acld, and is found ln certainprotelns. 3. Amino ocidspossesstwo functional groupsnamely carboxyl(-CooH) qnd amlno (-NH). ln the physiologlcolsystem,they existas dipolar ionscommonly relerred to os zwitteriois. 4. Besidesthe 20 standard omino acidspresent in proteins, there are seuerolnon-stondard amino ocids.Theseinclude the omino acid deriuatluesfound in proteins(e.g. hydroxy- prollne, hydroxylysine)ond, non-proteinamino acids(e.g. ornithine, citrulltne). 5. The structure of protein is dluided into t'our leuels of organizatlon. The primary structure representsthe linear sequenceof amino ocids. The twisting ond spatial arrangement of polypepttde chdin is the secondary structure. Tertiary structure constltutesthe three dimensional structure of a t'unctional protein. The assemblyof slmilar or dtssimilar polypepilde subunils comprlses quaternary structure. 5. The determlnation of primary structureof a protein inuoluesthe knowledgeol quality, quantity and the sequenceof amino acidsin the polypeptide. Chemicaland enzymatic methods are employed t'or the determinotion of primary structure. 7. The secondarystructureof protein mainly conslstsof a-helix anilor ftsheet.a-Helix is stabiltzedby extensivehydrogen bondlng.ftPleated sheetis composedol two or more segmentsof fully extended polypepttde choins. 8. The tertlory and quaternary structuresol protetn are stabiltzedby non-coualent bonds such os hydrogen bonds,hydrophobic interactions, ionic bondsetc. 9' Protelnsare classilied lnto three major groups. Simple proteins contain only amino acid resldues (e.g. albumtn). Conjugated proteins contaln a non-protein moiety known os prosthetic group, bestdesthe amtno acids (e,g. glycoprotelns). Dertued protelns are obtained by degradation of simple or conjugoted protelns. 10. In oddition to proteins, seueral peptldes perlorm btologtcolly tmportant functtons. These lnclude glutothlone, oxgtocin and udsopressin.
  • 68 BIOCHEMISTRY I. Essayquestions 1. Describethe classificationof aminoacidsalongwith their structures. 2. Discussthe organizationof proteinstructure.Cive an accountof the determinationof primary structureof orotein. 3. Describethe classificationof proteinswith suitableexamples. 4. Write an accountof non-standardamino acids. 5. Discussthe importantbiologicallyactivepeptides. II. Short notes (a) Essentialamino acids,(b) Zwitterion,(c) Peptidebond, (d) Edman'sreagent,(e) a-Helix, (fl p-Pleatedsheet,(g)Denaturation,(h) lsoelectricpoint,(i) Clutathione,(j) Quaternarystructure of protein. IIL Fill in the blanks 1. The averagenitrogencontentof proteins 2. Proteinsare the polymersof -. 3. Namethe sulfurcontainingessentialamino acid 4. The chargedmoleculewhich is electricallyneutralis knownas-. 5. The non -o aminoacid presentin coenzymeA -. 6. The bondsformingthe backboneof proteinstructure 7. The amino acid that is completelydestroyedby acid hydrolysisof protein L The numberof peptidebondspresentin a decapeptide-. 9. The chemicalnameof Sanger'sreagent 10. The phenomenonof disorganizationof nativeproteinstructureis knownas-. IV.Multiple choice questions 11,The iminoacidfoundin proteinstructure (a)Arginine(b) Proline(c) Histidine(d) Lysine, 12, Thefollowingis a non-proteinaminoacid (a)Ornithine(b) Homocysteine(c) Histamine(d)All of them. 13. The bondsin proteinstructurethat are not brokenon denaturation. (a)Hydrogenbonds(b) Peptidebonds(c) lonic bond (d) Disulfidebonds. 14. Sequenatorisan automaticmachineto determineaminoacidsequencein a polypeptidechain. The reagentusedin sequenatoris (a)Sanger'sreagent(b) CNBr (c)Trypsin(d) Edman'sreaBent, 15. The reactiongivenby two or morepeptidelinkagesis (a)Biurettest(b) Ninhydrintest(c)Xanthoproteicreaction(d) Pauley'stest.
  • nNucXefcAcids andnNucXeotfldes J here are two types of nucleic acids, I namely deoxyrihonucleic acid (DNA) and ribonucleicacid (RNA).Primarily,nucleic acids serveas repositoriesand transmittersof genetic information. Brief history DNA was discoveredin 1869 by Johann Friedrich Miescher, a Swiss researcher.The demonstrationthat DNA contained genetic informationwas first made in 1944, by Avery, Macleodand MacCarv. Functions of nucleic acids DNA is the chemical basisof heredityand nay be regardedas the reservebank of genetic rrormation.DNA is exclusivelyresponsiblefor -aintaining the identity of differentspeciesof c'ganismsover millionsof years.Further,every asoectof cellularfunctionis underthe controlof f,{. The DNA is organized into geneg the :urdamental units of genetic information. The genescontrol the protein synthesisthrough the mediationof RNA, as shown below A----+ RNA-----| Prlteir The interrelationshipof thesethreeclassesof biomolecules(DNA,RNAandproteins)constitutes the cenfral dogma of molecular biology or more commonly the central dogma of life. Components of nucleie aeids Nucleic acids are the polymers of nucleotides (polynucleotides)held by 3' and 5' phosphate bridges.In other words, nucleic acids are built up by the monomericunits-nucleotides(lt may be recalledthat protein is a polymer of amino acids). Nucleotidesare composedof a nitrogenous base,a pentosesugarand a phosphate.Nucleo- tidesperforma wide varietyof functionsin the livingcells,besidesbeingthebuildingblocksor 69
  • 70 BIOCHEMISTRY Flg.5.l : Generalstructureofnitrogenbases (A)Purine(B) Pyrimidine(Th€ pasltionsarenambercd awrding to theintemetif'.'d'lsystern). monomericunits in the nucleicacid (DNA and RNA) structure. These include their role as structuralcomponentsof some coenzymesof B-complexvitamins(e.9. FAD, NAD+), in the energy reactionsof cells (ATP is the energy currency), and in the control of metabolic reactions. STRUCTUREOF NUCLEOTIDES As alreadystated,the nucleotideessentially consistsof nucleobase,sugar and phosphate. Thetermnucleosiderefersto base+ sugar.Thus, nucleotideis nucleoside+ phosphate. Purines and pyrinnidines The nitrogenousbasesfound in nucleotides (and, therefore,nucleic acids)are aromatic heterocycliccompounds.The basesare of two types-purinesand pyrimidines.Theirgeneral structuresare depictedin Fig.S.l. Purinesare numberedin the anticlockwisedirectionwhile pyrimidinesare numberedin the clockwise direction.Andthisisan internationallyaccepted systemto representthe structureof bases. Major bases in nucleie acids The structuresof major purines and pyrimidinesfoundin nucleicacidsareshownin Fig.5.2.DNAandRNAcontainthesamepurines namelyadenine(A) and guanine(C). Further, thepyrimidinecytosine(C)isfoundin bothDNA and RNA. However,the nucleicacidsdiffer with respectto the secondpyrimidinebase. DNA contains thymine (T) whereas RNA containsuracil (U). As is observedin the Fig,5.2,thymineanduracildifferin structureby thepresence(inT)or absence(inU)of a methyl 8roup. Tautomeric fcrms of purines an€i pyriffiidines The existenceof a molecule in a keto (lactam) and enol (lactim) form is known as tautomerism.Theheterocyclicringsof purines /o and pyrimidineswith oto LC-/ functional groupsexhibittautomerismassimplifiedbelow. OH l. ^1, I'v-r- .i- OH I -C=N- Lactamform Lactlmform o H Adenlne(A) (6-aminopurine) Guanlne(G) (2-amino6-orypurine) I Cytoslne(C) (2-ory4-aminopyrimidine) oo H3 HN" tI lll f*/ Hil Thymine(T) Uracil(U) (2,A-dioxy-Smethylpyrimidine)(2,4-dioxypyrimidine) Flg.5.2: Structuresofmajorpurines(A,G)and pyrimidlnes(C, T,U)foundin nucleicacids.
  • Ghapter 5 : NUCLEICACIDSAND NUCLEOTIDES NHe t- a N2-c llli/- ^ 6"'"*"-" I H NHe t- N?cc lll ,t-' HO D-Ribose OHH D-2-Deoryribose Fig. 5.3 : The tautomeric forms of cytosine. Fig. 5.5: Structuresof sugarspresentin nucleicacids (riboseis foundin RNAanddeoxyribosein DNA;Note theslructuraldifferenceat Cz). Lactamform Lactimform The purine-guanine and pyrimidines- cytosine,thymineand uracilexhibittautomerism. The lactam and lactim forms of cytosine are representedin Fi9.5.3. At physiologicalpH, the lactam(keto)tauto- meric forms are predominantlypresent. Minor basesfound in nucleicacids : Besides the basesdescribedabove, severalminor and unusualbasesareoftenfound in DNA and RNA. These include 5-methylcytosine, Na-acetyl- cytosine, N6-methyladenine,N6, N0-dimethyl- adenine,pseudouraciletc. lt is believedthat the unusualbasesin nucleicacidswill help in the recognitionof specificenzymes. Other biologically important bases : The basessuch as hypoxanthine,xanthineand uric acid (Fig.5.4)are presentin the free state in the cells. The former two are the intermediatesrn purine synthesiswhile uric acid is the end product of purine degradation. Purinebasesof plants: Plantscontaincertain methylated purines which are of pharmaco- logical interest. These include caffeine (of coffee),theophylline (of tea) and theobromine (of cocoa). Sugars of nucleic acids The five carbon monosaccharides(pentoses) are found in the nucleic acid structure.RNA contains D-ribose while DNA contains D-deoxyrihose.Riboseand deoxyribosediffer in structureat C2. Deoxyribosehasone oxygenless at C2 compared to ribose (Fig.s.A. Nomenclattrre of nueleotides The addition of a pentose sugar to base produces a nucleoside.lf the sugar is ribose, ribonucleosides are formed. Adenosine, guanosine, cytidine and uridine are the ribonucleosidesof A, C, C and U respectively.lf the sugar is a deoxyribose, deoxyribo- nucleosidesare produced. The term mononucleotide is used when a single phosphate moiety is added to a nucleoside. Thus adenosine monophosphate (AMP)containsadenine+ ribose+ phosphate. The principal bases, their respective nucleosidesand nucleotides found in the structureof nucleicacidsare given in Tahle5.1. Note that the prefix 'd' is usedto indicateif the sugaris deoxyribose(e.g.dAMP). The binding of nucleotide components The atoms in the purine ring are numbered as .l to 9 and for pyrimidineas 1 to 6 (SeeFig.S.l).The carbonsof sugarsare representedwith an associated prime (1 for differentiation. Thus the pentose carbonsare 1' to 5'. Hypoxanthine Xanthine Uricacid (6-orypurine) (2,6-diorypurine) (2,6,8-trioxypurine) Fig. 5.4 : Structures of some biologically impoftant purines.
  • 72 BIOCHEMISTFIY Ribonucleoside Ribonucleotide (5'-monophosphate) Abbreviation Adenine(A) Guanine(G) Cytosine(C) Uracil(U) Adenosine Guanosine Cytidine Uridine Adenosine5'-monophosphateoradenylate Guanosine5'-monophosphateorguanylate CytidineSamonophosphateorcytidylate Uridine5'-monophosphateoruridylate AMP GMP CMP UMP Deoxyribonucleoside Deoxyribonucleotide (5'-monophosphate) Abbreviation Adenine(A) Guanine(G) Cytosine(C) Thymine(T) Deoxyadenosine Deoxyguanosine Deorycytidine Deoxythymidine Deoxyadenosine5'-monophosphateordeoryadenylate DeoryguanosineSlmonophosphateordeoxyguanylate Deorycytidine5'-monophosphateordeoxycytidylate Deoxythymidine5'-monophosphateordeoxythymidylate dAMP dGMP dCMP dTMP The pentosesare bound to nitrogenousbases by p-N-glycosidicbonds.The Ne of a purinering binds with C1111of a pentosesugarto form a covalentbond in the purine nucleoside.ln case of pyrimidinenucleosides,the glycosidiclinkage is between Nl of a pyrimidine and C'1 of a pentose. The hydroxyl groups of adenosine are esterifiedwith phosphatesto produce 5'- or 3'-monophosphates.5'-Hydroxyl is the most commonlyesterified,hence5' is usuallyomitted while writing nucleotide names. Thus AMP represents adenosine 5'-monophosphate. However, for adenosine3'-monophosphate,the abbreviation3'-AMP is used. The structuresof two selected nucleotioes namefyAMP and TMP are depictedin Fig.5.6. H{urr:"freosidedi- and triphosphates Nucleoside monophosphatespossessonly one phosphatemoiety(AMP,TMP).The addition of secondor third phosphatesto the nucleoside resultsin nucleosidediphosphate(e.g.ADP) or triphosphate(e.9.ATP),respectively. *o o--P-o-H2? o-[ c- P-o-H2g l AMP OHH TMP Fig.5.6 : Thestructuresof adenosineS'-monophosphate(AMP)and thymidineS'-monophosphate(TMP) [*-Addition of second or third phosphate gives adenosine diphosphate (ADP) and adenosine triphosphate (ATP) respectivetyl.
  • Chapter 5 : NUCLEICACIDSAND NUCLEOTIDES 73 Fig. 5.7 : Structures of selected purine and pyimidine analogs. The anionic propertiesof nucleotidesand nucleic acids are due to the negativecharges contributedby phosphategroups. PUR|NE, PYRIMTDTNE AND NUCLEOTIDEANALOGS It is possible to alter heterocyclic ring or sugar moiety, and produce synthetic analogs of purines, pyrimidines, nucleosides and nucleotides.Some of the syntheticanalogsare highlyusefulin clinicalmedicine.The structures of selectedpurine and pyrimidine analogsare given in Fi9.5.7. The pharmacologicalapplicationsof certain analogsare listedbelow 1. Allopurinol is used in the treatment of hyperuricemia and gout (For details, Refer Chapter l7). 2. S-Fluorouracil, 6-mercaptopurine, 8-aza- guanine, 3-deoxyuridine,5- or 6-azauridine, 5- or 6-azacytidineand 5-idouracilareemployed in the treatmentof cancers.Thesecompounds get incorporated into DNA and block cell oroliferation. 3. Azathioprine (which gets degraded to 6-mercaptopurine) is used to suppress immunologicalrejectionduring transplantation. 4. Arabinosyladenine is used for the treatment of neurological disease, viral enceohalitis. 5. Arabinosylcytosineis beingusedin cancer therapyas it interfereswith DNA replication. 6. The drugs employed in the treatmentof AfDS namely zidovudine or AZT (3-azido 2',3'-dideoxythymidine) and didanosine (dideoxy- inosine)are sugar modified synthetic nucleotide analogs(Fortheir structureand moredetailsRefer Chapter 3A. DNA is a polymer of deoxyribonucleotides (or simply deoxynucleotides).lt is composedof monomeric units namely deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxy- cytidylate(dCMP)and deoxythymidylate(dTMP) (lt may be noted herethat some authorspreferto useTMP for deoxythymidylate,since it is found only in DNA). The details of the nucleotide structureare given above. Schematic representation of polynucleotides The monomericdeoxvnucleotidesin DNA are hefd together by 3',5'-phosphodiesterbridges (Fi9.5.81. DNA (or RNA) structure is often representedin a short-handform. The horizontal line indicatesthe carbon chain of sugar with base attachedto C,,. Near the middle of the horizontalIine is C3,phosphatelinkagewhile at the otherend of the line is C5,phosphatelinkage (Fig.s.A. Ghargaff's rule of DNA composltion Erwin Chargaff in late 1940s quantitatively analysedthe DNA hydrolysatesfrom different species.He observedthat in all the specieshe studied,DNA hadequalnumbersof adenineand thymine residues(A = T) and equal numbersof guanineand cytosineresidues(G = C). This is known as Chargaff'srule of molar equivalence between the purines and pyrimidines in DNA structure.The significanceof Chargaff'srule was not immediatelv realised.The double helical structure of DNA derives its strength from Chargaff'srule (discussedlater). 8-Azaguanine
  • 74 BIOCHEMISTFIY 'end I I 5' J I o I I o L 2q' I {K H ,: I Hzr 4 DNA structure is considered as a milestone in the era of modern biology. The structure of DNA double helix is comparableto a twisted ladder. The salient features of Watson-Crick model of DNA (now known as B-Df.lA) are described next (Fi9.5.9). A ____T A____ | u ==:: u T ---- A J = =:='J Fig.5.8: Structureofa polydeoryribonucleotide segmentheldbyphosphodiesterbonds.Onthelower partis therepresentationol shorthad formof oligonucleotides. Single-strandedDNA, and RNAs which are usually single-stranded,do not obey Chargaff's rule. However, double-strandedRNA which is the genetic material in certain virusessatisfies Chargaff'srule. DNA DOUBLEHELIX The double helical structureof DNA was proposed by lames Watson and FrancisCrick in 1953 (Nobel Prize, 1962).The elucidationof uanrne OH I n-D-/l- C 3'end A Fig.5.9 : (A) Watson-Crickmodel of DNA helix (B) Complementary base pairing in DNA helix. 1'
  • Ghapter 5 : NUCLEICACIDSAND NUCLEOTIDES 1. The DNA is a righthandeddoublehelix.lt consists ol two polydeoxyribonucleotide chains (strands) twisted around each other on a common axts. 2. The two strands are antiparallel, i.e., one strand runs in the 5' to 3' direction while the otherin 3'to 5'direction.Thisis comparableto two parallel adjacent roads carrying traffic in oppositedirection. 3. The width (or diameter)of a double helix is 20 Ao (2 nm). 4. Each turn (pitch) of the helix is 34 A" (3.4 nm) with 10 pairsof nucleotides,each pair placed at a distanceof about 3.4 Ao. 5. Each strand of DNA has a hydrophilic deoxyribosephosphatebackbone(3'-5'phospho- diesterbonds)on the outside(periphery)of the molecule while the hydrophobic bases are stackedinside(core). 6. The two polynucleotide chains are not identicalbut complementaryto each other due to basepairing. 7. The two strands are held together by hydrogen bonds formed by complementary base pairs (Fig.S.|O).The A-T pair has 2 hydrogen bonds while G-C pair has 3 hydrogen bonds.The G = C is strongerby about50% than A=T. 8. The hydrogenbonds are formed betweena purine and a pyrimidineonly. lf two purines face each other, they would not fit into the allowable space. And two pyrimidineswould be too far to form hydrogen bonds. The only base arrangementpossible in DNA structure, from spatialconsiderationsis A-T, T-A, G-C and c-c. 9. The complementarybase pairing in DNA helix proves Chargaffs rule. The content of adenineequalsto that of thymine (A = T) and guanineequalsto that of cytosine(G = C). 10. The genetic information resideson one of the two strands known as template strand or sensestrand.The opposite strand is antisense strand. The double helix has (wide) major groovesand (narrow)minor groovesalong the phosphodiesterbackbone.Proteinsinteractwith DNA at these grooves,without disruptingthe basepairs and double helix. Sonformations 0f DNA double helEx Variation in the conformation of the nucleotides of DNA is associated with conformationalvariants of DNA. The double helical structureof DNA exists in at least 6 differentforms-A to E and Z. Among these,B, A and Z forms are important (Table 5.2). The B-form of DNA double helix, described bv Watsonand Crick (discussedabove),is the most predominant form under physiological conditions. Eachturn of the B-form has 10 base pairsspanninga distanceof 3.4 nm. The width of the double helix is 2 nm. The A-form is also a right-handedhelix. lt contains11 basepairsperturn.Thereis a tilting of the base pairs by 2O" away from the central axts. The Z-form (Z-DNA) is a left-handedhelix and contains '12 base pairs per turn. The Fiq.5.10 : Complementarybasepaiing in DNA (A) Thymine pairs with adenine by 2 hydrogen bonds (B) Cytosine pairs with guanine by 3 hydrogen bonds. H (B) .Z-=.,-N.-,, l' tl n.. ttt'o
  • 76 BIOCHEMISTFIY Feature B-DNA A-DNA Z.DNA Helixtype Right-handedRight-handedLetl-handed Helical diameter(nm) Distanceper eachcomplete turn(nm) Riseperbase pair(nm) Numberol base pairspercomplete rurn Basepairtilt +19" -1.2' (variable) HelixaxisrotationMajorgrooveThroughbase Minorgroove pairs(variable) polynucleotide strands of DNA move in a somewhat 'zig zag' fashion, hence the name Z-DNA. It is believedthat transition between different helical forms of DNA playsa significantrole in regulatinggeneexpression. OTHER TYPES OF DNA STRUCTURE It is now recognized that besides double helical structure, DNA also exists in certain unusual structures. lt is believed that such structures are important for molecular recognitionof DNA by proteinsand enzymes. This is in fact neededfor the DNA to discharge its functions in an appropriatemanner. Some selected unusual structures of DNA are brieflv described. Eent DNA In general,adenine base containing DNA tractsare rigidand straight.Bentconformationof DNA occurswhen A-tractsare replacedby other basesor a collapseof the helix into the minor grooveof A-tract.Bendingin DNA structurehas also been reported due to photochemical damageor mispairingof bases. Certain antitumor drugs (e.g. cisplatin) produce bent structurein DNA. Such changed structurecan take up proteins that damage the DNA. Triple-stranded DNA Triple-strandedDNA formation may occur due to additional hydrogenbonds betweenthe bases.Thus,a thymine can selectivelyform two Hoogsteen hydrogen bonds to the adenine of A-T pair to form I-A-L Likewise, a protonated cytosinecan alsoform two hydrogenbondswith guanineof C-C pairsthat resultsin C-G-C. An outline of Hoogsteentriple helix is depicted in Fig.5.11. Triple-helical structure is less stable than doublehelix.Thisis due to the factthatthe three negativelycharged backbone strands in triple helix results in an increased electrostatic repulsion. Four-stranded DNA l Polynucleotideswith very high contents of guanine can form a novel tetramericstructure 1.84 0.370.290.34 -9" tzl110 Fig" 5.11: An outlineof Hoogsteentriplehelical structureof DNA.
  • chapter 5 : NUCLEICACIDSAND NUCLEOTIDES 77 called G-quarfefs. These structuresare planar and are connected by Hoogsteen hydrogen bonds (Fig.S.12A).Antiparallel four-stranded DNA structures, referred to as G-tetraplexes have also been reported Gig.5.12Rl. The ends of eukarvoticchromosomesnamely telomeresare rich in guanine,and thereforeform C-tetraplexes.In recent years, telomereshave becomethe targetsfor anticancerchemotherapies. Fig.5.12: Four-strandedDNAstructure(A)Parallel C-tetraplexeshave been implicated in the recombinationof immunoglobulingenes,and in dimerization of double-strandedgenomic RNA of the human immunodeficiencyvirus (Hlu. THE SIZE OF DNA MOLECULE -UNITS OF LENGTH DNA moleculesare huge in size. On an average, a pair of B-DNA with a thickness of 0.34 nm has a molecular weight of 660 daltons. Forthe measurementof lengths,DNA double- strandedstructureis considered,and expresssed in the form of base pairs (bp). A kilobasepair (kb) is 103 bp, and a megahasepair (Mb) is 106 bp and a gigabasepair (Cb) is 10e bp. The kb, Mb and Cb relationsmav be summarizedas follows : 1 kb = 1000 bp 1 Mb = 1000 kb = 1,000,000bp 1 Cb = 1000 Mb = 1,000,000,000bp It may be noted herethat the lengthsof RNA molecules (like DNA molecules)cannot be expressedin bp, since most of the RNAs are single-stranded. The length of DNA varies from speciesto species,and is usuallyexpressedin termsof base pair composition and contour length. Contour lengthrepresentsthe total lengthof the genomic DNA in a cell.Someexamplesof organismswith bp and contour lengthsare listed. . l, phage virus- 4.8 x 104 bp-contour length16.5 mm. E. coli - 4.6 x 106 bp - contour length 1.5 mm. Diploid human cell (46 chromosomes)- 6.0 x 10ebp-contour length2 meters. It may be notedthatthe genomicDNA sizeis usually much larger the size of the cell or nucleuscontainingit. Forinstance,in humans,a 2-meter long DNA is packed compactly in a nucleusof about 1Opmdiameter. (B) ?-fG-G ?-?G_G IItt t-l3' s', / f_l I G_G tl tl tl G_G tltl 3' s', s', Iu G I u I r: I G4uartets (B)AntiparallelG-tetraplex.
  • 78 BIOCHEMISTtrIY The genomic DNA rnay exist in linear or circular forms. Most DNAs in bacteria exist as closed circles. This includes the DNA of bacterial chromosomes and the extra- chromosomalDNA of plasmids.Mitochondria an<lchloroplastsof eukaryoticcellsalsocontain circularDNA. ChromosomalDNAs in higherorganismsare mostly linear. indiiziduaihurnanchromosomes contain a sinqle DN,t r-noleculewith variable sizes compactly packed. Thus the smallest chromosomecontains34 Mb while the larsesr one has263 Mb. ffiffiWATUffiAT[ON MF MN& STffiANffiS The tr,vo strandsof DNA helix are held together by hydrogen bonds. Disruption of hycJrogenbonds(by changein pl-1or increasein iemperature) results in the separation of polynucleotidestrancls.Thisphenonrenon of Joss of helical structure of DNA is kno',vn as denaturatian (Fi9.5.13). The phosp[64;"r*"t bonds are not broken by denaturation.Lossof helical structurecan be measuredby increase in absorbance at 260 nm iin a sDectro- photometer). Denaturation. - R"a"tr-rti* Twostrands separated Fiq.5.13 : Diagrammaticrepresentationof denaturation and renaturationof DNA. Melting temperature (Im) is defined as the temperatureat which halfof the helicalstructure of DNA is lost.SinceC-C basepairsare more stable(due to 3 hydrogenbonds)than A-T base pairs(2 hydrogenbonds),the Tm is greaterfor DNAs ivith higherC-C content.Thus,the Tm is 65"C for 35% C-C contentwhile it is 70'C for 5A% C-C content. Formanride destabilizes hydrogenbonds of base pairs and, therefore, lowersTrn.Thischemicalcompoundiseffectivell, usedin recombinantDNA experiments. EIOMEDICAL/ CLINTCALCONCEPTS L€ D/VA is the reseruebank of geneticint'ormation,ultimately responsiblet'or the chemical bcsiso/ life and heredity. Gt DNA is organized into genes, the t'undamental units of genetic int'ormation. Genes control protein biosynfhesisthrough the mediationof RNA. u-FNucleic acidsare the polymers of nucleotides.Certoin nucleotidesserueas B-complex uitamin coenzymes(EAD'IVAD+,CoA),carriersot' highenergyintermediates(UDP-glucose, S-adenosylmethionine)and secondmessengersol hormonal action (cAME cGMP). Na Uric ocid is a purine, ond the end product ol purine metabolism,that has been implicatedin the disordergout. Certain purine basesfrom plants such os caft'eine(oj coffee),theophylline(of tea)and theobromine(of cocoo)are of pharmacologicalinterest. Synthetic analogsof bases(5-fluorouracil,6-mercaptopurine.6-azauridine)are used to inhibit the growth of cancercells. c'Ji' ut Certainantitumordrugs(e.g.cisplatin)can producebent DNA structureand damageit.
  • Ghapter 5 : NUCLEICACIDSAND NUCLEOTIDES 79 Renaturation(or reannealing)is the process in which the separatedcomplementary DNA strandscan form a double helix. As alreadvstated.the double-strandedDNA helix in each chromosomehas a lengththat is thousandstimesthe diameterof the nucleus.For instance,in humans,a 2-meter long DNA is packed in a nucleusof about 'l0 pm diameter! This is made possible by a compact and marvellouspackaging,and organizationof DNA insidein cell. OrganEzation of prakaryotFc Dl{A In prokaryoticcells,the DNA is organizedas a singlechromosomein the form of a double- strandedcircle. These bacterial chromosomes are packed in the form of nucleoids, by interaction with oroteins and certain cations (polyamines). Srganization of eukaryotic DhlA In the eukaryoticcells,the DNA is associated with various proteins to form chromatin which then gets organized info compact structures namely chromosomes(Fig.SJa" The DNA doublehelix iswrappedaroundthe core proteinsnamely histoneswhich are basicin nature.The core is composedof two molecules of histones(H2A, H2B, H3 and H4). Eachcore with two turns of DNA wrapped round it (approximatelywith 150 bp) is termed as a nucleosome, the basic unit of chromatin. Nucleosomesare separatedby spacer DNA to which histone H1 is attached (Fig.S.l5).This continuousstring of nucleosomes,representing beads-on-astringform of chromatinis termedas 10 nm fiber. The length of the DNA is considerablyreducedby the formationof 10 nm fiber. This 10-nm fiber is further coiled to oroduce 30-nm fiber which has a solenoid structurewith six nucleosomesin everv turn. These 30-nm fibers are further organized into loops by anchoringthe fiber at M-rich regions namely scaffold-associatedregions(SARS)to a proteinscafold.Duringthe courseof mitosis,the loops are further coiled, the chromosomes condenseand becomevisible. RNA is a polymer of ribonucleotidesheld together by 3',5'-phosphodiester bridges. AlthoughRNA hascertainsimilaritieswith DNA structure,they have specificdifferences l. Pentose: The sugarin RNA is ribosein contrastto deoxyribosein DNA. 2. Pyrimidine: RNA containsthe pyrimidine uracil in placeof thymine(in DNA). 3. Singlestrand : RNA is usuallya single- strandedpolynucleotide.However, this strand may fold at certain placesto give a double- strandedstructure,if complementarybasepairs are in closeproximity. 4. Chargaff's rule-not obeyed : Due to the single-strandednature, there is no specific relation between purine and pyrimidine contents.Thusthe guaninecontentis not equal to cytosine(asis the casein DNA). 5. Susceptibilityto alkali hydrolysis: Alkali can hydrolyseRNA to 2',3'-cyclicdiesters.This is possibledue to the presenceof a hydroxyl group at 2' position.DNA cannot be subjected to alkali hydrolysisdue to lack of this group. 6. Orcinol colour reaction : RNAs can be histologically identified by orcinol colour reactiondue to the presenceof ribose. TYPES OF RNA The three major types of RNAs with their respectivecellularcompositionare givenbelow 1. MessengerRNA (mRNA) : 5-1O"/" 2. TransferRNA (IRNA) : 10-200/" 3. RibosomalRNA (rRNA) : 50-80%
  • 80 BIOCHEMISTF|Y Jzn'NakedDNA doublehelix I,.", | ,o*'', 'Beads-on-a-string' formofchromatin 30-nmchromatin fibrecomposedof nucteosomes Chromosomeinan extendedform (non-condensed loops) Condensedform ol cnromosome Metaphase chromosome Fig.5.l4 : OrganizationofeukaryoticDNAstructurein theformofchromatinandchromosomes. +I 11nm lnbmucleosome Fig.5.15: Structurcofnucleosomes.
  • Ghapter 5 : NUCLEICACIDSAND NUCLEOTIDES 81 Typeof RNA Abbreviation Funclion(s) MessengerRNA Transfersgenelicintormationfromgenesto Ii99.9_0,.T.99.!9_9J.$ltgli'e.p$giLL ...lt_qt:r.qg:l.:.qtE..tlf9l99.t..EllL TransferRNA ServesasprecursorformRNAandotherRNAs. TransfersaminoacidtomRNAforprotein biosynthesis. RibosomalRNA rRNA TMRNA Providesstructuralframeworkforribosomes. SmallnuclearRNA snRNA Involvedin mRNAprocessing. SmallnucleolarRNA snoBNA Playsa keyroleintheprocessingofrRNA molecules. SmallcytoplasmicRNA scRNA Involvedintheselectionoforoteinsforexoort. Mostlypresentinbacteria.Addsshortpeptide tagst0proteinstofacilitatethedegradationof incorrectlysynthesizedproteins. Transfer-messengerRNA Besidesthe three RNAs referredabove, other RNAsarealsopresentin the cells.Theseinclude heterogeneousnuclear RNA (hnRNA), small nuclear RNA (snRNA),small nucleolar RNA (snoRNA)and small cytoplasmicRNA (scRNA). The major functionsof theseRNAsare given in Table 5.3. The RNAsaresynthesizedfrom DNA, and are primarily involved in the process of protein biosynthesis (Chapter 2fl. The RNAs vary in their structureand function. A brief description on the major RNAs is given. Messenger RNA (mRNAl The mRNA is synthesizedin the nucleus(in eukaryotes) as heterogeneous nuclear RNA (hnRNA). hnRNA, on processing,liberatesthe functionalmRNA which entersthe cytoplasmto participate in protein synthesis.mRNA has high molecularweight with a short half-life. The eukaryotic mRNA is capped at the S'-terminal end by 7-methylguanosine triphosphate.lt is believedthat this cap helpsto prevent the hydrolysis of mRNA by 5'-exo- nucleases.Further,the cap may be also involved in the recognitionof mRNAfor proteinsynthesis. The 3'-terminal end of mRNA contains a polymer of adenylate residues (20-250 nucleotides) which is known as poly (A) tail. Thistail may providestabilityto mRNA, besides preventingit from the attack of 3'-exonucleases. mRNA molecules often contai certain modified basessuch as 6-methyladenylatesin the internalstructure. Transfer RNA (tRNAl Transfer RNA (soluhle RNA) molecule contains 71-80 nucleotides(mostly 75) with a molecularweight of about 25,000.Thereare at least20 speciesof tRNAs,correspondingto 20 amino acids present in protein structure.The structure of tRNA (for alanine) was first elucidatedby Holley. The structure of IRNA, depicted in Fig.S.t6, resemblesthat of a clover leaf. IRNA contains mainly four arms,each arm with a basepaired stem. 1. The acceptor arm : This arm is capped with a sequenceCCA (5'to 3'). The amino acid is attachedto the acceptor arm. 2. The anticodon arm : This arm, with the three specific nucleotide bases(anticodon),is responsiblefor the recognitionof triplet codon of mRNA. The codon and anticodon are complementaryto each other. t
  • 82 BIOCHEMISTRY Anticodonarm Fiq.5.16: Structureof transferRNA. 3. The D arm : lt is so named due to the presenceof dihydrouridine. 4. The TYC arm : This arm contains a sequenceof T, pseudouridine(representedby psi,Y) and C. 5. The variable arm : This arm is the most variable in tRNA. Based on this variabiliW, tRNAs are classifiedinto 2 categories: (a) Class I tRNAs : The most predominant (about 75"/") form with 3-5 base pairs length" (b) Classll tRNAs : They contain 13-20 base pair long arm. Basepairs in tRNA : The structureof IRNA is maintained due to the complementary base pairing in the arms. The four arms with their respectivebasepairsare given below The acceptorarm - 7 bp The TYC arm - 5 bp The anticodonarm - 5 bp TheDarm -4bp f,ibosomal RNA (rRNAl The ribosomes are the factories of protein synthesis. The eukaryotic ribosomes are composed of two major nucleoprotein complexes-60Ssubunit and 40S subunit. The 605 subunit contains28S rRNA, 55 rRNA and 5.8SrRNA while the 40S subunitcontains18S rRNA.The functionof rRNAsin ribosomesis not clearly known. lt is believed that they play a significantrole in the binding of mRNA to ribosomesand protein synthesis. Other RNAs The various other RNAs and their functions are summarisedin Table5.3. CATALYTIG RNAs-RI BOZYMES In certaininstances,the RNA componentof a ribonucleoprotein (RNA irr association with protein) is catalyticallyactive. Such RNAs are termedasribozymes.At leastfive distinctspecies of RNAthat act ascatalystshavebeenidentified. Three are involved in the self processing reactions of RNAs while the other two are regarded as true catalysts (RNase P and rRNA). Ribonuclease P (RNase P) is a ribozyme containing protein and RNA component. lt cleaves IRNA precursorsto generate mature tRNA molecules. RNA molecules are known to adapt tertiarystructurejust like proteins(i.e.enzymes). The specific conformation of RNA may be responsiblefor its function as biocatalyst.lt is believed that ribozymes (RNAs) were functioning as catalysts before the occurrence of protein enzymes, during the course of evolution. el g-Amino acid I +A -!, r Acceptorarm Darm Complementary basepairs TrCarm Variablearm
  • Shapten 5 : NUCLEICACIDS AND NUCLEOTIDES 83 l. 2. 3. 4. 5. 6. 7. 8. 9. 10. DNA is the chemical basisof heredity organizedinto genes,the basicunits of genetic inJormotion. RNAs ImFNA, fRNA and rRNA) are produced by DNA which in turn carry out protein synfhesis. Nucleic acids are the polymers of nucleotides (polynucleoiides)held by 3' and 5' phosphodiesterbridges. A nucleotide essentiolly consists of base + sugdr (nucleoside) and phosphate. Besidesbeing the constituentsof nucleicacid structurc,nucleotidesperform a wide uariety ot' cellulor functians (e.9.energycarriers,metabolicregulators,second messengersetc.) Both DNA ond RNA containthe parines-adenine(A)and guanlne(G)ond thepyrimidine- cytosine(C).The secondpgrimidine is thymine (TJin DNA while it is uracil (U) in RNA. The pentose sugar,D-deoxyriboseis lound in DNA while it is D-ribosein RNA. The structure o/ DNA is a double helix (Watson-Crickmodel) composed ol two antiparollel sfrondsol polydeoxynucleotidestutistedaround eachothen The strandsare held together by 2 or 3 hydrogen bondst'ormed betweenthe basesi.e. A = T: G : C. DNA sfructure satisfiesChargoff'srule that the content of A is equal to T, and that ol G equal to C. Besidesthe double helical structure, DNA olso exisfs fn certain unusuol structures- bent DNA, triple-strand DNA, four-strand DNA. RNA is usually a singlestranded polyribonucleotide.mRNA is capped ot 5'terminol end by 7-methylGTPwhile at the 3'-terminal end, it contains o poly A toil. mRNA speciliesthe sequenceol amino scids in protein synfhesis. The structure of tRNA resembles that of a clouer leaf with four arms (acceptori anticodon, D-,and T'LC)held by complementargbasepoirs. fRNA deliuersamino acids lor protein synthesis. Certain RNAs that mn function os enzymes are termed os ribozymes. Ribozymes were probablVfunctioningoscofolysfsbeforethe occurrenceof protein enzymesduring ewlution.
  • 84 BIOCHEMISTFIY I. Essayquestions 1. Describethe structureof DNA. 2. NamedifferentRNAsand discusstheir structure. 3. Writean accountof structure,functionand nomenclatureof nucleotides. 4. Describethe structureof nitrogenousbasespresentin nucleicacids.Add a noteon tautomerism. 5. "The backboneof nucleicacid structureis 3'-5'phosphodiesterbridge."-justify. II. Short notes (a) Chargaff'srule, (b) Riboseand deoxyribose,(c) Hydrogenbonds in DNA, (d) Nucleoside, (e) Differentformsof DNA, (f)TransferRNA, (g) Purinebasesof plants,(h) Complementarybase pairs,(i) DNA denaturation,(j) hnRNA. III. Fill in the blanks Thefundamentalunit of geneticinformationis known as DNA controlsproteinsynthesisthroughthe mediationof Nucleicacidsare the polymersof The pyrimidinepresentin DNA but absentin RNA Riboseand deoxvribosedifferin theirstructurearoundcarbonatom Nucleotideis composedof The scientistwho observedthatthereexistsa relationshipbetweenthe contentsof purinesand pyrimidinesin DNA structure(A = T; C = C) B. The basepair G-C is morestableand strongerthanA-T due to 9. Underphysiologicalcondition,the DNA structureis predominantlyin the form 10. The acceptorarm of IRNA containsa cappednucleotidesequence IV. Multiple choice questions 1'l. The nitrogenousbasenot presentin DNA structure (a)Adenine(b) Cuanine(c) Cytosine(d) Uracil. 12. The numberof basepairspresentin eachturn (pitch)of B-formof DNA helix (a)e (b) 10 (c) 11 (d) 12. 13. The backboneof nucleicacid structureis constructedby (a)Peptidebonds(b) Glycosidicbonds(c) Phosphodiesterbridges(d)All of them. 14. The followingcoenzymeis a nucleotide (a)FAD(b) NAD+ (c) CoASH(d)All of them. 15. The nucleotidethat servesas an intermediatefor biosyntheticreaction (a)UDP-glucose(b)CDP-acylglycerol(c) S-Adenosylmethionin8(d)AII of them. 1. 2. 3. 4. 5. 6. 7.
  • The etrzytnes spetrr. : "Weare the catalystsof the liuing worU! Protein in nature, and in d.ctionspecifc, rapid and accurate; Huge in size but with srnall actiaecentres; Highly erploitedfor diseasediagnosisin hb cennel" f nzymesare biocatalysts- the catalystsof life. L A catalvsf is defined as a subsfance that increasesihe velocity or rate of a chemical reactionwithout itselfundergoingany changein the overall process. The student-teacherrelationshipmay be a ' good example to understandhow a catalyst works.The studentsoftenfind it difficultto learn from a text-book on their own. The teacher explainsthe subjectto the studentsand increases their understandingcapability.lt is no wonder that certain difficult things which the students takedaystogetherto understand,and sometimes do not understandat all - are easilylearntunder the guidanceof the teacher.Here, the teacher acts like a catalyst in enhancing the understandingabilityof students.A goodteacher is alwaysa good catalystin students'life! Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in nature (exception - RNA acting as ribozyme), colloidal and thermolahile in character, and specific in their action. In the laboratory,hydrolysisof proteinsby a strongacid at 100'C takesat leasta couple of days.The same protein is fully digestedby the enzymes in gastrointestinaltract at body temperature(37'C) within a couple of hours. This remarkable difference in the chemical reactionstaking place in the living system is exclusivelydue to enzymes.The very existence of life is unimaginablewithout the presenceof enzvmes. Berzeliusin 1836 coined the term catalysis (Greek: to dissolve).In 1878, Kuhneusedthe word enzyme (Creek: in yeast)to indicatethe catalysistaking place in the biologicalsystems. lsolationof enzyme systemfrom cell-freeextract of yeastwas achievedin 1883 by Buchner.He named the active principle as zymase (later found to contain a mixture of enzymes),which could convertsugarto alcohol. ln 1926, James 85
  • 86 BIOCHEMISTF}Y Sumner first achieved the isolation and crystallizationof the enzyme ureasefrom jack bean and identifiedit as a protein. In the early days, the enzymeswere given names by their discoverers in an arbitrary manner.Forexample,the namespepsin,trypsin and chymotrypsinconvey no informationabout the function of the enzymeor the natureof the substrateon which they act. Sometimes,the suffix-asewas addedto the substratefor naming the enzymese.g. lipaseactson lipids; nuclease on nucleic acids; lactaseon lactose.Theseare known as trivial namesof the enzymes which, however, fail to give complete informationof enzyme reaction (type of reaction, cofactor requirementetc.) Enzymesaresometimesconsideredundertwo broad categories: (a) Intracellular enzymes- They are functionalwithin cells where they are synthesized.(b) Extracellularenzymes- These enzymes are active outside the cell; all the digestiveenzymesbelong to this group. The InternationalUnionof Biochemistry(lUB) appointedan EnzymeCommissionin 1961.This committeemadea thoroughstudyof the existing enzymesand devisedsome basic principlesfor the classificationand nomenclatureof enzymes. Since 1964, the IUB system of enzyme classification has been in force. Enzymes are divided into six major classes(in that order). Eachclasson itsown representsthe generaltype of reactionbrought about by the enzymesof that class(Table6.1. Enzymeclasswith examples* Reactioncatalysed 1. Oxidoreductases Alcoholdehydrogenase(alcohol: NAD*oxidoreductaseE.C., cytochromeoxidase,L-andD-aminoacidoxidases 2. Transferases Hexokinase(ATP: D-hexose6-phosphotransferase,E.C., transaminases,transmethylases,phosphorylase Hydrolases Lipase(triacylglycerolacylhydrolaseE.C.,choline esterase,acidandalkalinephosphatases,pepsin,urease 4. Lyases Aldolase(ketose1-phosphatealdehydelyase,E.C., fumarase,histidase Oxidation------+Reduction AHr+B->A+BH, Grouptransfer A-X+ B------+A+ B-X 3. Hydrolysis A- B+ H"O------+AH+ BOH Addition------+Elimination A-B+i-v--Ax-Bi 5. lsomerases 6. Ligases Triosephosphaleisomerase(D-glyceraldehyde3-phosphate ketoisomerase,E.C.,retinolisomerase, phosphohexoseisomerase lnterconversionofisomers A-> A' Glutaminesynthetase(L-glutamateammonialigase,E.C., acetylCoAcarboxylase,succinatethiokinase Condensation(usuallydependentonATP) A+B;z-5-+A-B ATP ADp+pi *For oneenzynein eachclass,systenaticn"r, ttorg*,rh E.C. nunbetis givenin thebrackets.
  • Chapter 6 : ENZYMES 1. Oxidoreductases: Enzymesinvolved in oxidation-reductionreactions. 2. Transferases: Enzymesthat catalysethe transferof functionalgroups. 3. Hydrolases : Enzymesthat bring about hydrolysisof variouscompounds. 4. Lyases : Enzymes specialised in the additionor removalof water,ammonia,COr etc. 5. lsomerases: Enzymesinvolved isornerizationreactions. 6. ligases: Enzymescatalysingthe synthetic :eactions(Creek : ligate-to bind) where two nroleculesare joined togetherand ATP is used. lThe word OTHLIL (firstletter in each class) rray be memorisedto rememberthe six classes of enzymesin the correctorderl. Eachclassin turn is subdividedinto manv sub-classeswhich are further divided. A four digit Enzyme Commission (EC.) number is assignedto each enzyme representingthe class (firstdigit),sub-class(seconddigit),sub-subclass (third digit) and the individualenzyme (fourth digit). Each enzyme is given a specific name indicatingthe substrate,coenzyme(if any) and the typeof the reactioncatalysedby the enzyme. Although the IUB namesfor the enzymes are specificand unambiguous,they have not been acceptedfor general use as they are complex and cumbersometo remember.Therefore,the trivial names,along with the E.C. numbersas and when needed, are commonly used and widely accepted. All the enzymesare invariablyproteins.In recent years, however, a few RNA molecules have been shown to function as enzvmes.Each enzymehasitsown tertiarystructureand specific conformation which is very essentialfor its catalytic activity. The functional unit of the enzyme is known as holoenzymewhich is often made up of apoenzyme (the protein part) and a coenzyme (non-protein organic part). Holoenzyme -----+Apoenzyme + Coenzyme (activeenzyme) (proteinpart) (non-proteinpart) The term prostheticgroup is used when the non-protein moiety tightly (covalently) binds with the apoenzyme. The coenzyme can be separatedby dialysisfrom the enzymewhile the prostheticgroup cannot be. The word monomeric enzyme is used if it is made up of a single polypeptidee.g. ribo- nuclease,trypsin.Someof the enzymeswhich possessmore than one polypeptide (subunit) chain are known as oligomeric enzymes e.g. lactate dehydrogenase, aspartate trans- carbamoylaseetc. Thereare certainmultienzyme complexespossessingspecific sites to catalyse differentreactionsin a sequence.Only the native intactmultienzymecomplexisfunctionallyactive and not the individualunits,if they areseparated e.g.pyruvatedehydrogenase,fattyacid synthase, prostaglandinsynthaseetc. The enzymesexhibit all the generalpropertiesof proteins(Chapter4). Genetic engineering and modified enzymes Recentadvancesin biotechnologyhavemade it possibleto modify the enzymeswith desirable characters-improvedcatalyticabilities,activities under unusual conditions. This approach is required since enzymes possess enormous potentialfor their use in medicineand industry. Hybrid enzymes: lt is possibleto rearrange genesand produce fusion proteins.e.g. a hybrid enzyme (of glucanaseand cellulase)that can more efficiently hydrolysebarley p-glucansin beer manufacture. Site-directed mutagenesis : This is a techniqueusedto producea specifiedmutation at a predeterminedpositionin a DNA molecule. The result is incorporationof a desiredamino acid (of one's choice) in place of the specified amino acid in the enzyme.By this approach,it is possibleto producean enzymewith desirable characteristics.e.g. tissueplasminogenactivator (used to lyse blood clots in myocardial the
  • 88 BIOCHEMISTF|Y Fig. 6.1 : Effect of enzyme concentration on enzyme velociy. infarction) with increased half-life. This is achieved by replacing asparagine(at position 120) by glutamine. ln recentyears,it hasalsobecomepossibleto produce hybrid enzymes by rearrangementof genes. Another innovative approach is the production of abzymesor catalytic antibodies, the antibody enzymes. The contact between the enzyme and substrateis the most essentialpre-requisitefor enzyme activity. The important factors that influencethe velocityof the enzymereactionare discussedhereunder t. Goncentration of enzyme As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases(Fig.6.l). In fact, this property of enzyme is made use in determining the serumenzymesfor the diagnosisof diseases. By usinga known volumeof serum,and keeping all the other factors (substrate,pH, temperature etc.)at the optimum level,the enzymecould be assayedin the laboratory. 2" Concentration of substrate Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels.A rectangularhyperbolais obtainedwhen velocity is plotted against the substrate concentration(Fig.6.2).Threedistinct phasesof the reactionare observedin the graph(A-linear; B-curve;C-almostunchanged). Order of reaction : When the velocity of the reactionis almost proportionalto the substrate concentration(i.e.[S] is lessthan Kn,),the rateof the reaction is said to be first order with respect to substrate.When the ISJis much greaterthan Kn', the rate of reaction is independent of substrateconcentration,and the reactionis said to be zero order. Enzymekinetics and K, value : The enzyme (E)and substrate(S)combine with each other to form an unstableenzyme-substratecomplex (ES) for the formationof product (P). k1. E+Sr,-- rsSr + p 'k- Here kl , k2 and k3 represent the velocity constants for the respective reactions, as indicatedby arrows. K' the Michaelis-Mentenconstant(or Brigrs and Haldane's constant),is given by the formula K' = kz+kr k, The following equation is obtained after suitablealgebraicmanipulation. v = Vr"* [S] equation(1) Km+[S] where v = Measuredvelocity, Vtu" S K. = Maximumvelocitv, = Substrateconcentration, = Michaelis- Mentenconstant. + I I II o -9o o E N t! Letus assumethatthe measuredvelocity(v) isequalto f Vrr". Thentheequation(1)maybe substitutedasfollows 1v _ v.axlsl 2.max K.+[s]
  • Ghapter 6 : ENZYMES 89 Vr"* -J 1V.", TI II -F g Substrateconcentration--+ since V."" is approached asymptotically.By taking the reciprocalsof the equation (1), a straightline graphic representationis obtained. r^1 1_ Km .. 1 [".| ---1- -----------i--TV v-"" [s] v'"-[sJ 1- Km., 1 1- = -,.-i--Ti- V V..,. IS.| Vr"* The above equationis similarto y = ax + b. Therefore,a plot of the reciprocalof the velocity I ' I ur. the reciprocalof the substrateconcen- vi / _ tration l--l--| givesa straightline. Herethe slope tsl /- is K./y'.u* and whose y intercept is 1/y'."*. The Lineweaver-Burk plot is shown in Fig.6.3. lt is much easierto calculatethe K. from the intercepton x-axis which is -(l/Km). Further,the double reciprocalplot is useful in understandingthe effect of various inhibitions (discussedlater). Enzyme reactions with two or more substrates: The above discussionis basedon the presumptionof a single substrate-enzyme reaction. In fact, a majority of the enzyme- catalvsed reactions involve two or more substrates. Even in case of multisubstrate _1 Km 1 tsl ! Fig. 6,2 : Effect of substrate concentration on enzyme velocity (A-linear; B-curve; C-almost unchanged). Km+ [S] - 2vmax[S] Vr"* Kn'+[S] = 2[S] Km = [S] K stands for a constant and m stands for Michaelis(in Kn.). K^ or lhe Michaelis-Menten constant is defined as the substrate concentration (expressedin moles/l)to produce half-maximum velocity in an enzyme catalysed reaction. lt indicatesthat half of the enzyme molecules(i.e. 50%) are bound with the substratemolecules when the substrateconcentrationequalsthe K. value. K. value is a constantand a characteristic feature of a given enzyme (comparableto a thumb impression or signature). lt is a represe;;rtativefor measuringthe strengthof ES complex. A low K^ value indicates a strong affinity between enzyme and substrate,whereas a high K. value reflectsa weak affinity between them. For majority of enzymes,the K. values are in the rangeof 10-s to 10-2 moles. lt may however,be notedthat K. is not dependenton the concentrationof enzvme. Lineweaver-Burkdouble reciprocalplot : For the determinationof K, value, the substrate saturationcurve (Fig.5.2)is not very accurate Ftg,6.3 : Lineweaver-Burkdoublereciprocalplot,
  • 90 BIOCHEMISTRY enzymes, despite the complex mathematical expressions,the fundamentalprinciplesconform to Michaelis-MentenKinetics. 3. Effect of temperature Velocityof an enzymereactionincreaseswith increasein temperatureup to a maximum and then declines. A bell-shapedcurve is usually observed (Fig"6.a). Ternperaturecoefficient or Qto is defined as increase in enzyme velocity when the temperatureis increasedby 10"C.Fora majority af enzymes, Qlo is 2 between 0"C and 40oC. lncrease in temperature results in higher activation energy of the molecules and more moiecuiar(enzymeand substrate)collision and interaction for the reaction to oroceed faster. The optimum temperaturefor most of the enzymesis between40'C-45'C. However, a few enzymes (e.g. venom phosphokinases,muscle adenylatekinase)areactiveevenat 100'C. Some plant enzymeslike ureasehaveoptimum activity around 60'C. This may be due to very stable structureand conformationof theseenzymes. In general,when the enzymesare exposedto a temperature above 50"C, denaturation leading to derangementin the native (tertiary)structure of the proteinand activesite are seen.Majority of the enzymes become inactive at higher temperature(above70'C). 20 30 40 50 60 Temperature("C) It is worth noting herethat the enzymeshave beenassignedoptimaltemperaturesbasedon the laboratory work. These temperatures,however, may have less relevance and biological significancein the living system. 4. Effect of pH lncreasein the hydrogen ion concentration (pH)considerablyinfluencesthe enzymeactivity and a bell-shaped curve is normally obtained (Fig.6.5).Eachenzyme has an optimum pH at which the velocity is maximum. Below and above this pH, the enzyme activity is much lower and at extremepH, the enzyme becomes totally inactive. Most of the enzymes of higher organisms show optimum activityaround neutralpl-l (6-8). Thereare,however,manyexceptionslike pepsin (1-2), acid phosphatase(4-5) and alkaline phosphatase(10-X1).Enzyrnesfrom fungi and plantsare most active in acidic pH (a-6). Hydrogenions influencethe enzyme activity by alteringthe ionic chargeson the aminoacids (particularly at the active site), substrate,ES complex etc. 5, Effect of product concentration The accumulation of reaction products generally decreases the enzyme velocity. I o q) E N UI Io o) o E N ttl Fig" 6.4 : Ef'fect of Iempenture on enzyme velocity.
  • Chapter 6 : ENZYMES Forcertainenzymes,the productscombinewith the active site of enzyme and form a loose complexand, thus, inhibitthe enzymeactivity. ln the living system,this type of inhibition is generally prevented by a quick removal of productsformed.The end product inhibition by feedbackmechanismis discussedlater. 6" Effect of activators Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+, zn2+, ca2+, co2*, cu2+, Na+, K+ etc" for their optimum aciivity"Rarely,anionsarealsoneeded for enzyme activity e.g. chloride ion (C11 for amylase. Metals function as activatorsof enzymevelocitythroughvariousmechanisms- combining with the substrate,formation of ES-metalcomplex, direct participation in the reactionand bringing a conformationalchange in the enzyme. Two categoriesof enzymesrequiring metals fbr their activity are distinguished . Metal-activated enzymes : The metal is not tightly held by the enzyme and can be exchangedeasilywith other ions e.g. ATPase(Mg2*and Ca2*) Enolase(Mg2*) " Metalloenzymes : These enzymes hold the metals rather tightly which are not readily exchanged. e.g.. alcohol dehydro- genase,carbonic anhydrase,alkaline phos- phatase, carboxypeptidase and aldolase containzinc. Phenoloxidase(copper); Pyruvateoxidase(manganese); Xanthineoxidase(molybdenum); Cytochromeoxidase(iron and copper). 7. Effect of time Under idealand optimalconditions(likepH, iemperature etc.), the time required for an enzymereactionis less.Variationsin the time of the reaction are generally related to the alterationsin pH and temperature. Fig. 6.6 : A diagrammatic representationof an enzyme with active site. L Effect of light and raEliation Exposureof enzymes to ultraviolet, beta, gamma and X-raysinactivatescertain enzyrnes due to the formationof peroxides.e.g. UV rays inhibit salivaryamylaseactivity. Enzymes are big in size compared to substrateswhich arerelativelysmaller.Evidently, a smallportionof the hugeenzymemoleculeis directly involved in the substratebinding and caialysis(Fig.6,6). The active site (or active centre) of an enzynte representsas the small region at wkich tke suhstrate(s) binds and participates in the aatalysis. Salient features of active site 1. The existenceof active site is due to the tertiary structureof protein resulting in three- dimensionalnativeconformation. 2. The activesite is made up of amino acids (known as catalytic residues)which are far fronr eachotherin the linearsequenceof aminoacids (primarystructLrreof protein).For instance,the enzyme lysozyme has 129 amino acids. The activesiteisformedby the contributionof amino acid residuesnumbered35, 52, 62, 63 and 101. 3. Active sites are regarded as ctrefts or crevicesor pocketsoccupyinga small region in a big enzymemolecule. Activesite
  • 92 BIOCHEMISTFIY 4. The activesite is not rigid in structureand shape.lt is ratherflexible to promotethe specific substratebinding. 5. Cenerally, the active site possessesa substrate binding sife and a catalytic site. The latteris for the catalysisof the specificreaction. 6. The coenzymes or cofactors on which some enzymesdepend are presentas a part of the catalyticsite. 7. The substrate(s)binds at the activesite by weak noncovalentbonds. 8. Enzymesare specificin theirfunctiondue to the existenceof active sites. 9. The commonlyfound amino acidsat the active sites are serine, aspartate, histidine, cysteine,lysine,arginine,glutamate,tyrosineetc. Among these amino acids, serine is the most frequentlyfound. 10. The substratelslbinds the enzyme (E)at the activesiteto form enzyme-substratecomplex (ES).The product(P)is releasedafterthe catalysis and the enzymeis availablefor reuse. il:+:1-i.' Enzyme inhibitor is defined as a substance which bindswith the enzymeand bringsabout a decreasein catalyrtc activity of that enzyme. The inhibitor may be organic or inorganicin nature. There are three broad categoriesof enzymeinhibition 1. Reversibleinhibition. 2. Irreversibleinhibition. 3. Allostericinhibition. l. Reversible inhibition 1 ,' The inhibiior binds non-covalently with enzyme and the enzyme inhibition can be reversed if the inhibitor is removed. The reversibleinhibitionis furthersub-dividedinto l. Competitiveinhibition (Fig.6.7A) ll. Non-competitiveinhibition (Fig.6.7B) Fig. 6.7 : A diagrammaticrepresentationof (A) Competitive and (B) Non-competitiveinhibition. C=Substrate Non-competitive inhibitor Enzyme-inhibitor complex Enzyme-inhibitor comprex l. Competitive inhibition : The inhibitor (l) which closelyresemblesthe real substrate(S)is regardedas a substrateanalogue.The inhibitor competeswith substrateand binds at the active site of the enzyme but does not undergo any catalysis.As long as the competitive inhibitor holdsthe activesite,the enzymeis not available for the substrateto bind. Duringthe reaction,ES and El complexesare formed as shown below ES--+E+ P EI The relativeconcentrationof the substrateano inhibitor and their respectiveaffinity with the enzyme determinesthe degree of competitive inhibition.The inhibitioncould be overcomebv a high substrateconcentration.ln competitive inhibition, the K- value increaseswhereas V-u* remains unchanged (Fig.6.A. The enzyme succinatedehydrogenase(SDH) is a classicalexampleof competitiveinhibition with succinic acid as its substrate. The compounds,namely,malonicacid,glutaricacid and oxalic acid, have structuralsimilaritywith succinicacid and competewith the substratefor bindingat the activesiteof SDH. P Active site
  • chaprer 6 : ENZYMES 93 cH2cooH cH2cooH Succinic acid cooH I CHr t- cooH Malonic acid Methanol is toxic to the body when it is converted to formaldehyde by the enzyme alcohol dehydrogenase(ADH). Ethanol can competewith methanolfor ADH. Thus,ethanol can be used in the treatment of methanol porsonrnS. Some more examplesof the enzymeswith substratesand competitiveinhibitors(of clinical and pharmacologicalsignificance)are given in Table 6.2. Antimetabolites : These are the chemical compoundsthat block the metabolic reactions by their inhibitory ,action 'on enzymes. Antimetabolitesare usuallystructuralanalogues of substratesand thus are competitiveinhibitors (Table6.2).They are in usefor cancertherapy, Bout etc. The term antivitamins is used for the antimetaboliteswhich block the biochemical actions of vitamins causing deficiencies,e.g. sulphonilamide,dicumarol. ll. Non-competitiveinhibition : The inhibitor binds at a site other than the active site on the enzyme surface. This binding impairs the enzymefunction.The inhibitorhasno structural resemblancewith the substrate.However,there usuallyexistsa strongaffinityfor the inhibitorto bind at the secondsite.In fact,the inhibitordoes not interferewith the enzyme-substratebinding. But the catalysisis prevented,possiblydue to a distortionin the enzyme conformation. The inhibitor generally binds with the enzyme as well as the EScomplex.The overall relation in non-competitive inhibition is representedbelow E+sirES-*E+P ++ I .lf I tfEI+S EIS For non-competitiveinhibition,the K^ value is unchangedwhile V^^* is lowered (Fig.5.9). Heavy metal ions (Ag+,Pb2+,Hg2+etc.)can non-competitively inhibit the enzymes by binding with cysteinyl sulfhydryl groups. The generalreactionfor Hg2+ is shown below. E-SH + Hgt*i^ E-S. . .Hg2++ H+ I f/ 2'mu + II Km,Km 1 tsl (B) 11 Km Km' (A) Fig.6.8 : Effect of competitive inhibitor (i) on enzyme velocity. (A) Velocity (v) versus substrate (S) plot. (B) Lineweaver-Burk ptot (Red lines with inhibitor; campetitive inhibitor increases K^, unalters V^o).
  • 94 BIOCHEMISTRY Enzyme Substrate lnhibitor(s) Significanceof inhibitor(s) Xanthineoxidase Hypoxanthine xanthine Allopurinol Usedinthecontrolofgouttoreduceexcess productionofuricacidfromhypoxanthine. Monoamineoxidase Catecholamines (epinephdne,norepinephrine) Usefulforelevatingcatecholaminelevels.Ephedrine, amphetamine DihvdrofolatereductaseDihvdrofolicacid Aminopterin, amethopterin, methotrexate Employedinthetreatmentofleukemiaand othercancers. AcetylcholineesteraseAcetylcholine SuccinylcholineUsedinsurgerylormusclerelaxation,in anaesthetisedpatients. Dihydropteroate synthase Paraaminobenzoicacid (PABA) Sulfonilamide Preventsbacterialsynthesisoffolicacid. VitaminK epoxide VitaminK Dicumarol Actsasananticoagulant. reductase HMGCoAreductase HMGCoA Lovastatin, compactin Inhibitcholesterolbiosynthesis Heavv metalsalso lead to the formation of covalent bonds with carboxyl groups and histidine,oftenresultingin irreversibleinhibition. 2. lrreversible inhibition The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible.These inhibitorsare usuallvtoxrc poisonoussubstances. lodoacetateis an irreversibleinhibitorof the enzymes like papain and glyceraldehyde 3-phosphatedehydrogenase.lodoacetatecombines with sulfhydryl(-SH) groupsat the activesiteof theseenzvmesand makesthem inactive. 71/ 2 vmax T
  • Ghapter 6 : ENZYMES Diisopropyl fluorophosphafe (DFP) is a nerve gas developed by the Cermans during Second World War. DFPirreversiblybindswith enzymes containingserineat the active site, e.g. serine proteases, acetylcholine esterase. Many organophosphorusinsecticides like melathionare toxic to animals(includingman) as they block the activity of acetylcholine esterase (essential for nerve conduction), resultingin paralysisof vital body functions. Disulfiram (Antabuse@)is a drug used in the treatmentof alcoholism. lt irreversiblvinhibits the enzyme aldehyde dehydrogenase.Alcohol addicts, when treated with disulfiram become sick due to the accumulationof acetaldehyde, leadingto alcohol avoidance.(Nofe ; Alcohol is metabolizedby two enzymes. lt is first acted upon by alcohol dehydrogenase to yield acetaldehyde.The enzyme aldehyde dehydro- genaseconvertsacetaldehydeto acetic acid.) The penicillin antibioticsact as irreversible inhibitors of serine- containing enzymes, and block the bacterialcell wall svnthesis. lrreversibleinhibitorsare frequentlyused to identifyamino acid residuesat the activesiteof the enzymes, and also to understand the mechanismof enzyme action. Snicide inhibition Suicide inhibition is a specializedform of irreversibleinhibition.ln this case,the original inhibitor (the structural analogue/competitive inhibitor)is convertedto a more potentform by the sameenzymethat oughtto be inhibited.The so formed inhibitor binds irreversiblywith the enzyme. This is in contrast to the original inhibitorwhich binds reversibly. A good example of suicide inhibition is allopurinol (used in the treatmentof gout, Refer Chapter lV. Allopurinol, an inhibitor of xanthineoxidase,getsconvertedto alloxanthine, a more effectiveinhibitor of this enzyme. The use of certain purine and pyrimidine analoguesin cancertherapy is also explained on the basis suicide inhibition. For instance, S-fluorouracil gets converted to fluorodeoxy- uridylatewhich inhibitsthe enzymethymidylate synthase,and thus nucleotidesynthesis. 3. Allosteric inhibition The detailsof this type of inhibitionare given under allosteric regulation as a part of the regulation of enzyme activity in the living svstem. Enzymesare highly specific in their action when comparedwith the chemicalcatalysts.The occurrence of thousands of enzymes in the biological systemmight be due to the specific nature of enzymes. Three types of enzyme specificityare well-recognised 1. Stereospecificity. 2. Reactionspecificity, 3. Substratespecificity, Specificity is a characteristic property of the active site. 1. Stereospecificity or optical specificity : Stereoisomersare the comoounds which have the same molecularformula,but differ in their structuralconfiguration. The enzymes act only on one isomer and, therefore,exhibit stereospecificity. e.g. L-amino acid oxidaseand D-amino acid oxidase act on L- and D-amino acids respectively. Hexokinaseacts on D-hexoses; Glucokinaseon D-glucose; Amylaseacts on a-glycosidiclinkages; Cellulasecleavesp-glycosidicbonds. Stereospecificityis explainedby considering three distinct regions of substrate molecule specificallybinding with three complementary regionson the surfaceof the enzyme(Fig.5.l0). The classof enzymesbelongingto isomerases do not exhihit stereospecificity, since they are specializedin the interconversionof isomers.
  • 96 BIOCHEMISTF|Y .l 2. Reaction specificity : The same substrate can undergo different types of reactions, each catalysed by a separateenzyme and this is referredto as reactionspecificity.An amino acid can undergo transamination,oxidative deami- nation, decarboxylation,racemizationetc. The enzymeshowever, are differentfor each of these reactions (For details, refer Chapter |fl. 3. Substrate specificity : The substrate specificityvariesfrom enzymeto enzyme.lt may be either absolute,relativeor broad. . Absolute substrate specificity : Certain enzymes act only on one substrate e.g. glucokinaseactson glucoseto give glucose6- phosphate,ureasecleavesurea to ammonia and carbon dioxide. . Relativesubstratespecificity : Someenzymes act on structurallyrelatedsubstances.This, in turn, may be dependenton the specificgroup or a bond present.The action of trypsin is a good example for group specificity (Refer Fig.8.7).Trypsin hydrolysespeptide linkage involving arginine or lysine. Chymotrypsin cleaves peptide bonds attached to aromatic amino acids (phenylalanine, tyrosine and tryptophan). Examples of hond specificity- glycosidasesacting on glycosidic bonds of carbohydrates,lipasescleavingesterbondsof lipidsetc. . Broad specificity : Some enzymes act on closely relatedsubstrateswhich is commonly known as broad substratespecificity, e.g. hexokinaseactson glucose,fructose/mannose and glucosamineand not on galactose.lt is possible that some structural similarity among the first four compounds makes them a common substratefor the enzyme hexokinase. The proteinpartof the enzyme,on itsown, is not alwaysadequateto bring aboutthe catalytic activity. Many enzymes require certain non- protein small additional factors, collectively referred to as cofactors for catalysis. The cofactorsmay be organic or inorganic in nature. The non-protein, organic, Iow molecular weight and dialysable substance associated with enzyme function is known as coenzyme. The functional enzyme is referred to as holoenzyme which is made up of a protein part (apoenzyme) and a non-protein part (coenzyme);The term prosthetic group is used when a non-proteinmoiety is tightly bound to the enzyme which is not easily separableby dialysis. The term activator is referred to the inorganiccofactor(like Ca2+,Mg2+,Mn2+ etc.; necessaryto enhanceenzyme activity. lt may, however, be noted that some authors make no distinction between the terms cofactor, coenzyme and prostheticgroup and use them interchangeably. Coenzymes are second substrates : Coenzymesare often regardedas the second substrates or co-substrafes, since thev have affinitywith the enzymecomparablewith that of the substrates.Coenzymesundergo alterations during the enzymaticreactions,which are later regenerated.This is in contrast to the substrate which is convertedto the product. Fig. 6.10 : Diagrammatic representation of sterco- specificity (a', b', Cl-three point attachment of
  • Chapter 6: ENZYMES 97 Coenzyme(abhreviation) Derived from vitamin Atom or group transferred Dependentenzyme (example) Thiaminepyrophosphate(TPP) Flavinmononucleotide(FMN) Flavinadeninedinucleolide(FAD) Thiamine Riboflavin Aldehydeorketo Transketolase Hydrogenandelectron L-Aminoacidoxidase Riboflavin Niacin D-Aminoacidoxidase Lactatedehydrogenase Lipoicacid Pyridoxine Pantothenicacid Acyl GlucoseSphosphatedehydrogenase Pyruvatedehydrogenasecomplex AlaninetransaminaseAminoorketo Thiokinase Folicacid ?ieeril giell. !9!n!c9{a1i1o_egqogngsv_lc9o!a1i1 _ _9o$t1ry * Detakforeachcoenzynearegivenin Chapter7onvitanins Onecarbon Formyltransferase (formyl,methenyletc.) CO, Pyruvatecarborylase Methyl/isomerisation MethylmalonylCoAmutase Coenzymesparticipate in various reactions involving transfer of atoms or groups like hydrogen,aldehyde,keto, amino, acyl, methyl, carbon dioxide etc. Coenzymesplay a decisive role in enzymefunction. Coenzymesfrom B-complex vitamins : Most of the coenzymesare the derivativesof water soluble B-complex vitamins. In fact, the biochemicalfunctionsof B-complexvitaminsare exertedthroughtheir respectivecoenzymes.The chapteron vitaminsgivesthe detailsof structure and function of the coenzymes(ChapterV. ln Table. 6.3, a summary of the vitamin related coenzymeswith their functionsis given. Non-vitamincoenzymes: Not all coenzymes are vitamin derivatives.There are some other organicsubstances,which have no relationwith vitaminsbut function as coenzymes.They rnay be consideredas non-vitamincoenzymese.g. ATP,CDP, UDP etc.The importantnon-vitamin coenzymesalong with their functionsare given in Tahle 6.4. Nucleotide coenzymes : Some of the coenzymespossessnitrogenousbase,sugarand Coenzyme Abbreviation Biochemicalfunctions Adenosinetriphosphate ATP CDP Donatesphosphate,adenosineandadenosinemonophosphate (AMP)moieties. Requiredinphospholipidsynthesisascarrierofcholineand ethanolamine. Cytidinediphosphate Uridinediphosphate UDP S-Adenosylmethionine (activemethionine) Phosphoadenosinephosphosulfate (activesulfate) Carrierofmonosaccharides(glucose,galactose),requiredlor $ycogensynthesis. SAM Donatesmethylgroupinbiosyntheticreactions. Donatessulfateforthesynthesisofmucopolysaccharides.
  • 9B BIOCHEMISTF|Y phosphate. Such coenzymes are, therefore, regarded as nucleotides e.g. NAD+, NADP+, FMN, FAD, coenzymeA, UDPC etc. Coenzymesdo not decide enzymespecificity : A particularcoenzymemayparticipatein catalytic reactions along with different enzymes. For instance,NAD+ acts as a coenzyme for lactate dehydrogenaseand alcohol dehydrogenase.ln both the enzymaticreactions,NAD+ is involved in hydrogen transfer. The specificity of the enzyme is mostly dependent on the apoenzyme and not on the coenzvme. Catalysisis the prime function of enzymes. The nature of catalysis taking place in the biological system is similar to that of non- biologicalcatalysis.Forany chemicalreactionto occur, the reactantshave to be in an activated stateor transitionstate. Enzymes lower activation energy : The energyrequiredby the reactantsto undergothe reaction is known as activation energy. The reactants when heated attain the activation energy. The catalyst (or the enzyme in the biologicalsystem)reducesthe activationenergy and this causesthe reaction to proceed at a lower temperature.Enzymesdo not alter the equilibrium constants,they only enhance the velocity of the reaction. The role of catalystor enzyme is comparable with a tunnelmadein a mountainto reducethe barrier as illustratedin Fig.6.l1. The enzyme lowers energy barrier of reactants, thereby making the reaction go faster. The enzymes reducethe activationenergyof the reactantsin such a way that all the biologicalsystemsoccur at body temperature(below 40"C). Enzyme.substrate complex formation The prime requisitefor enzyme catalysisis that the substrate(S) must combine with the enzyme (E) at the active site to form enzyme- substratecomplex (ES)which ultimately results in the productformation (P). E + S$ ES---+E+ P A few theorieshave beenput forth to explain mechanism of enzyme-substrate complex formation. Lock and key model or Fischer's template theory This theory was proposed by a Cerman biochemist,Emil Fischer.This is in fact the very first model proposed to explain an enzyme cataiysedreaction. According to this model, the structure or conformationof the enzymeis rigid.Thesubstrate fits to the bindingsite (now activesite)just as a key fits into the proper lock or a hand into the properglove.Thusthe activesiteof an enzymeis a rigid and pre-shapedtemplatewhere only a specificsubstratecan bind. This model does not giveany scopefor the flexiblenatureof enzymes, hencethe modeltotallyfailsto explainmanyfacts of enzymaticreactions,the mostimportantbeing the effectof allostericmodulators. Induced fit theory or Koshland's model Koshland, in 1958, proposed a more acceptable and realistic model for enzyme- substratecomplexformation.As per this model, +I I II lLl B Fig. 6.11 : Effect of enzyme on activation energy af a reaction (A is the substrate and I is the
  • Ghapter 6 : ENZYMES 99 Fig. 6.12 : Mechanism of enzyme-substrate(ES) MECHANISIII OF ENZYME GATATYSIS The formation of an enzyme-substrate complex(ES)is very crucialfor the catalysisto occur, and for the product formation. lt is estimated that an enzyme catalysed reaction proceeds106 to 1012times fasterthan a non- catalysedreaction.The enhancementin the rate of the reactionis mainly due to four processes: 1. Acid-basecatalysis; 2. Substratestrain; 3. Covalentcatalysis; 4. Entropy effects. 1. Acid-base catalysis : Role of acids and basesis quite importantin enzymology.At the physiologicalpH, histidineis the mostimportant amino acid, the protonated form of which functions as an acid and its corresponding conjugateas a base.The other acids are -OH group of tyrosine,-SH group of cysteine,and e-aminogroup of lysine.The conjugatesof these acids and carboxyl ions (COO-) function as bases. Ribonucleasewhich cleavesphosphodiester bondsin a pyrimidineloci in RNA is a classical example of the role of acid and base in the catalysis. 2. Substratestrain : lnductionof a strainon the substratefor ESformationis discussedabove. Duringthe courseof straininduction,the energy level of the substrateis raised, leading to a transitionstate. The mechanismof lysozyme(an enzyme of tears,that cleavesp-1,4glycosidicbonds)action is believed to be due to a combination of substratestrainand acid-basecatalysis. 3. Covalent catalysis : In the covalent catalysis,the negativelycharged (nucleophilic) or positivelycharged (electrophilic)group is presentat the active site of the enzyme. This group attacksthe substratethat resultsin the covalentbindingof the substrateto the enzyme. ln the serine proteases(so named due to the presence of serine at active site), covalent catalysisalong with acid-basecatalysisoccur, e.g. chymotrypsin,trypsin,thrombin etc. complex formdtion (A) Lock and key model (B) Induced fit theory G) Substrate strain theory. t the active site is not rigid and pre-shaped.The essentialfeaturesof the substratebindingsiteare presentat the nascentactivesite.The interaction of the substratewith the enzymeinducesa fit or a conformationchangein theenzyme,resultingin the formation of a strong substratebinding site. Further,due to inducedfit, the appropriateamino acidsof the enzymeare repositionedto form the activesiteand bringaboutthe catalysis(Fig.6.12). Inducedfit model hassufficientexperimental evidence from the X-ray diffraction studies. Koshland'smodel also explainsthe action of allostericmodulatorsand competitiveinhibition on enzymes. Substrate strain theory In this model,the substrateis straineddue to the inducedconformationchangein the enzyme. It is also possiblethat when a substratebindsto the preformedactivesite,the enzyme inducesa strain to the substrate.The strained substrate leadsto the formationof product. ln fact, a combination of the induced fit model with the substratestrainis consideredto be operativein the enzymaticaction.
  • 100 BIOCHEMISTFIY 4. Entropy effect : Entropy is a term used in thermodynamics.lt is definedas the extentof disorderin a system.The enzymesbringabouta decreasein the entropy of the reactants.This enables the reactantsto come closer to the enzyme and thus increasethe rate of reaction. In the actualcatalysisof the enzymes,more than one of the processes- acid-basecatalysis, substratestrain, covalent catalysisand entropy are simultaneouslyoperative.Thiswill help the substrate(s)to attaina transitionstateleadingto the formationof products. T}IERMODYNAMICS OF ENZYMATIC REACTIOITS The enzyme catalysed reactions may be broadly grouped into three types based on thermodynamic(energy)considerations. 1. lsothermic reactions : The energy exchange between reactantsand products is negligiblee.g.glycogenphosphorylase Clycogen+ Pi --+ Clucose1-phosphate 2. Exothermic(exergonic)reactions: Energy is liberatedin thesereactionse.s. urease Urea --+ NH3 + CO2 + energy 3. Endothermic (endergonic) reactions : Energy is consumed in these reactionse.g. glucokinase Clucose+ ATP------+Glucose6-phosphate+ ADP In biological system, regulationof enzyme activitiesoccurs at different stagesin one or more of the followingways to achievecellular economy. 1. Allostericregulation 2. Activationof latentenzymes 3. Compartmentationof metabolic pathways 4. Control of enzyme synthesis 5. Enzymedegradation 6. lsoenzymes i. &ilssterie regulation ;rnsl allo*terie inhlhition Someof the enzymespossessadditionalsites, known as allostericsites (Greek : allo-other), EIOMEDICAL/ CLINICALCONCEPTS s€ The existenceot' lit'eis unimaginablewithout the presenceol enzymes-the biocotalysts. se Majoritg of the coenzymes (TPP, NAD+, FAD, CoA) are deriued from B-complex uitamins in which t'orm the latter exert their biochemicalJunctions. 0s Competitiue inhibitors of certain enzymesare ot'great biological signit'icance.Allopurinol, emploged in the treatment of gout, inhibits xanthine oxidase to reduce the formation ot' uric acid.The other competitiueinhibitorsincludeaminopterin used in the treatment of cancers,sult'anilamideas antiboctericidalogent and dicumarol as on anticoagulant.. P- The nerue gas(diisopropyl t'luorophosphate), t'irstdeveloped by Germansduring Second World Wa4 tnhibits acetylcholineesterqse,the enzyme essentialfor nerve conduction and paralysesthe uital body functions. Many organophosphorus insecticides(e.9. melathion) also block the actiuityof acetylcholineesterase. te Penicillin antibioticsirreuersiblyinhibit serinecontqining enzymesof bacterialcell wall sunthesis.
  • Ghapter 6: ENZYMES 101 l" Fig. 6.13 : Diagrammaticrepresentationof an allastericenzyme(A) T-farm;(B] P-foffn; (C] Effectof aftag"eieffitW1.(D) Etleotaf altoqtide:ac,.4vaiw,. - besidesthe activesite.Suchenzymesare known as allostericenzymes.The allosteric sites are unique placeson the enzymemolecule. Allosteric effectors : Certain substances referredto as allosteric modulators (effectorsor modifiers)bind at the allostericsiteand regulate the enzyme activity. The enzyme activity is increasedwhen a positive(+) allostericeffector binds at the allostericsite known as activator site.On the other hand, a negative(-) allosteric effector binds at the allosteric site called inhibitorsiteand inhibitsthe enzymeactivity. Classesof allostericenzymes: Enzymesthat are regulated by allosteric mechanism are referred to as allosteric enzymes. They are divided into two classesbasedon the influence of allostericeffectoron K, and V.r*. . K-class of allosteric enzymes, the effector changesthe K. and not the V."". Double reciprocal plots, similar to competitive inhibition are obtained e.g. phospho- fructokinase. . V-classof allostericenzymes,the effectoralters the V.r* and not the Kr. Double reciprocal plots resemble that of non-competitive inhibitione.g. acetylCoA carboxylase. Conformational changes in allosteric enzymes : Most of the allostericenzymesare oligomeric in nature. The subunits may be identical or different. The non-covalent reversiblebindingof the effectormoleculeat the allosteric site brings about a conformational changein the activesiteof the enzyme,leading to the inhibition or activationof the catalytic activity (Fig.6.l3). In the concerted model, allostericenzvmesexist in two conformational states-the T (tenseor taut) and the R (relaxed). The T and R statesare in equilibrium. Allostericactivator(or)substrate Allostericinhibitor Allosteric inhibitors favour T state whereas activators and substratesfavour R state. The substratecan bind only with the R form of the enzyme.The concentrationof enzvmemolecule in the R state increasesas more substrateis added, thereforethe binding of the substrateto the allostericenzyme is said to be cooperative. Allostericenzymesgivea sigmoidalcurve(instead of hyperbola) when the velocity (v) versus substrate(S)concentrationare plotted (Fig.5.14). The term homotropic effect is used if the substrate influences the substrate binding through allosteric mechanism, their effect is always positive. Heterotropic effecf is used when an allostericmodulatoreffectsthe binding of substrate to the enzyme. Heterotropic interactions are either positive or negative. Selected examples of allosteric enzymes responsiblefor rapid control of biochemical pathways are given in Table 6.5. 1 o o E N E lrJ Hyperbolic Substrateconcentration------+ Fig. 6.14 : Effect of substrate concentration an allos-
  • l02 ElIOCHEMISTFIY Allosteric Enzyme Metabolicpathway Inhibitor Activator Hexokinase Phosphofructokinase lsocitratedehydrogenase Pyruvatecarborylase Fructose1,6-bisphosphatase CarbamoylphosphatesynthetaseI Tryptophanoxygenase AcetylCoAcarboxylase Glycolysis Glycolysis Krebscycle Gluconeogenesis Gluconeogenesis Ureacycle Tryptophanmetabolism Fattyacidsynthesis Glucose6-phosphate ATP AMP,ADP ADP,NAD- AcetylCoA N-Acetylglutamate L-Tryptophan lsocitrale ATP AMP Palmitale Feedback regillation The processol inhibiting the first step by the final product, in a series of enzyme catalysed reactionsof a metabolic pathway is referredto as feedback regulation. Look at the seriesof reactionsgiven below A el >B sp )C ,9€ rD 9a rE A is the initial substrate,B, C, and D are the intermediatesand E is the end product, in a pathway catalysed by four different enzymes (e1,e-2,e3,e4).Thevery firststep(A -+ B by the enzyme e1) is the most effective for regulating the pathway, by the final end product E. This type of control is often called negative feedback regulation since increasedlevelsof end product will resultin its (er) decreasedsynthesis.This is a real cellular economy to save the cell from the wasteful expenditure of synthesizing a compoundwhich is alreadyavailablewithin the cell. Feedbackinhibition or end product inhibition is a specialisedtype of allosteric inhibition necessaryto control metabolic pathways for efficientcellularfunction. Aspartate transcarbamoylase(ATCase) is a good example of an allosteric enzyme inhibited by a feedback mechanism. ATCase catalysesthe very first reaction in pyrimidine biosynthesis. Carbamoylphosphate+ Aspartate Feedback control Carbamoylaspartate+ Pi IY Cytidinetriphosphate(CTP) Carbamoylphosphateundergoesa sequence of reactionsfor synthesisof the end product, CTP. When CTP accumulates,it allosterically inhibitsthe enzymeaspartatetranscarbamoylase by a feedbackmechanism. Feedbackregulation or feedback inhibition? Sometimesa distinctionis made betweenthese two usages.Feedbackregulation representsa phenomenonwhile feedbackinhibition involves the mechanismof regulation.Thus, in a true sense,they are not synonymous.For instance, dietarycholesteroldecreaseshepaticcholesterol biosynthesisthrough feedback regulation.This does not involve feedback inhibition, since dietary cholesteroldoes not directly inhibit the regulatory enzyme HMG CoA reductase. However, the activity of gene encoding this enzyme is reduced(repression)by cholesterol. 2. Activation of latent enzymes Latentenzymes,as such, are inactive.Some enzymes are synthesized as Proenzymes or zymogenswhich undergo irreversiblecovalent
  • Ghapter 6 : ENZYMES 103 activation by the breakdown of one or more peptidebonds.Forinstance,proenzymes-namely chymotrypsinogen,pepsinogenand plasminogen, are respectively- convertedto the activeenzymes chymotrypsin,pepsinand plasmin. Certain enzvmes exist in the active and inactive forms which are interconvertible, depending on the needs of the body. The interconversion is brought about by the reversible covalent modifications, namely phosphorylation and dephosphorylation,and oxidationand reductionof disulfidebonds. Clycogenphosphorylaseis a muscleenzyme that breaks dow'n glycogen to provide energy. This enzyme is a homodimer (two identical subunits)andexistsin two interconvertibleforms. Phosphorylaseb (dephosphoenzyme)is inactive which is convertedby phosphorylationof serine residuesto active form phosphorylasea. The inactiveenzymephosphorylaseb is producedon dephosphorylationas illustratedbelow. Thereare someenzymeswhich are active in dephosphorylatedstate and become inactive when phosphorylatede.g. glycogen synthase, acetyl CoA carboxylase. A few enzymesareactiveonly with sulfhydryl (-SH) groups, €.8. succinate dehydrogenase, urease.Substanceslike glutathionebring about the stabilityof theseenzymes. E-S-S-E Oxidised E-SH + E-SH Reduced inactive 2G-SH GS-SG active E P P 3. Gompartnnentation Therearecertainsubstancesin the body (e.g., fatty acids,glycogen)which are synthesizedand alsodegraded.Thereis no point for simultaneous occurrenceof both the pathways.Cenerally,the synthetic (anabolic) and hreakdown (catabolic) pathways are operative in different cellular organellesto achieve maximum economy. For instance,enzymes for fatty acid synthesisare found in the cytosol whereas enzymes for fatty acid oxidation are presentin the mitochondria. Dependingon the needsof the body- through the mediationof hormonaland othercontrols- fatty acids are either synthesizedor oxidized. The intracellularlocationof certainenzymes and metabolicpathwaysis given in Table6.6. Phosphorylaseb Phosphorylasea (inactive). z (active) ' .,-_- phosphatase _./.-....-. ______----l /'E 2Pi Organelle Enzyme/metaboIic pathway Cytoplasm Aminotransferases;peptidases;glycolysis;hexosemonophosphateshunt;fattyacid synthesis;purineandpyrimidinecatabolism. Mitochondria Fattyacidoxidation;aminoacidoxidation;Krebscycle;ureasynthesis;electron transportchainandoxidativephosphorylation. Nucleus BiosynthesisofDNAandRNA. Endoplasmicreticulum(microsomes)Proteinbiosynthesis;triacylglycerolandphospholipidsynthesis;steroidsynthesisand reduction;cytochromeP4Eo;esterase. Lysosomes Lysozyme;phosphatases;phospholipases;hydrolases;proteases;lipases;nucleases. Golgiapparatus Glucose6-phosphatase;5'-nucleotidase;glucosyFandgalactosyl-transferases. Peroxisomes Catalase;urateoxidase;D-aminoacidoxidase;longchainfattyacidoxidation.
  • BIOCHEMISTRYl04 I I I ! { I 4" Control of enzyme synthesis Most of the enzymes, particularlythe rate limiting ones, are present in very low concentration.Nevertheless,the amount of the enzyme directly controls the velocity of the reaction, catalysed by that enzyme. Many rate Iimiting enzymeshave short half-lives.This helps in the efficientregulationof the enzyme levels. There are two types of enzymei-(a) Consti- tutive enzymes (house-keeping enzymes)-the levelsof which are not controlled and remain fairly constant. (b) Adaptive enzymes-their concentrationsincreaseor decreaseas per body needsand are well-regulated.The synthesisof enzymes (proteinsl is regulated by the genes (Refer Chapter 25). Inductionand repression: Theterm induction is used to represent increased synthesis of enzyme while repressionindicatesits decreased synthesis. Induction or repression which ultimatelydeterminesthe enzymeconcentration at the gene level through the mediation of hormonesor other substances. Examplesof enzymeinduction: The hormone insulin induces the synthesis of glycogen synthetase,glucokinase, phosphofructokinase and pyruvate kinase. All these enzymes are involved in the utilization of glucose. The hormonecortisolinducesthe synthesisof many enzymes e.B. pyruvate carboxylase,tryptophan oxygenaseand tyrosineaminotransferase. Examplesof repression: In many instances, substratecan repressthe synthesisof enzyme. Pyruvatecarboxylaseis a key enzyme in the synthesis of glucose from non-carbohydrate sourceslike pyruvateand amino acids.lf thereis sufficientglucoseavailable,thereis no necessity for its synthesis. This is achieved through repression of pyruvate carboxylase hy glucose. 5. Enzyme degradation Enzymesare not immortal,sinceit will create a seriesof problems.Thereis a lot of variability in the half-livesof individualenzymes.Forsome, it is in days while for others in hours or in minutes,e.g.LDHa- 5 to 6 days;LDHI - 8 to 12 hours;amvlase-3 to 5 hours. ln general,the key and regulatoryenzymes are most rapidly degraded.lf not needed,they immediately disappear and, as and when required,they are quickly sysnthesized.Though not alwaystrue, an enzymewith long half-lifeis usuallysluggishin its catalyticactivity. 6. lsoenzymes Multiple forms of the same enzymewill also help in the regulationof enzymeactivity,Many of the isoenzymesare tissue-specific.Although isoenzymesof a given enzymecatalysethe same reaction, they differ in K' V.nu*or both. e.g. isoenzvmesof LDH and CPK. Enzymesare neverexpressedin termsof their concentration (as mg or pg etc.), but are expressedonly as activities.Various methods have been introduced for the estimation of enzyme activities (particularlyfor the plasma enzymes). In fact, the activities have been expressedin many ways, like King-Armstrong units, Somogyi units, Reitman-Frankelunits, spectrophotometricunits etc. Katal In order to maintain uniformity in the expression of enzyme activities (as units) worldover,the EnzymeCommissionof IUB has suggestedradicalchanges.A new unit- namely katal(abbreviatedas kat)-was introduced.One kat denotes the conversion of one mole substrateper second(mol/sec).Activity may also be expressedas millikatals(mkat), microkatals (pkat)and so on. International Units (lUf Someworkerspreferto use standardunits or Sl units (SystemInternational).One Sl unit or InternationalUnit (lU) is definedas the amount of enzyme activity that catalysesthe conversion of one micromol of suhstrate per minute. Sl units and katal are interconvertible.
  • Chapter 6 : ENZYMES 1lU = (or) 1 nkatal = 60 pkatal 1.67 lU Laboratory use of enzyme units In the clinical laboratories,however, the units- namelv katalor Sl units-are vet to find a place.Many investigatorsstill usethe old units like King-Armstrongunits, Somogyi units etc. while expressingthe enzyme activities. lt is therefore,essentialthat the units of enzyme activity, along with the normal values, be invariablystatedwhile expressingthe enzymes for comparison. Ribozymes Ribozymesare a group ol ribonucleic acids thatfunctionasbiological catalysts,and they are regardedas non-proteinenzymes. Altman and his coworkers,in 1983, found that ribonucleaseP- an enzymetill then known to cleave precursorsof tRNAs to give tRNAs- was functional due to RNA componentpresent in the enzyme and not the protein part of the enzyme. The RNA part isolatedfrom ribonucleaseP exhibiteda trueenzymeactivityand alsoobeyed Michaelis-Mentenkinetics. Later studies have proved that RNA, in fact, can function as an enzyme and bring about the catalysis. RNA moleculesare known to adapta tertiary structurejust as in the case of proteins (i.e. enzymes).The specific conformation of RNA may be responsiblefor itsfunctionasbiocatalyst. It is believed that ribozymes IRNAs) were functioningas catalystsbeforethe occurrenceof proteinenzymesduring evolution. Certain enzymes agents, analytical manipulationsand (Table6.V. are useful as therapeutic reagents, in genetic for industrialapplications Enzyme Application Toremovebloodclots Incancertherapy Anti-inflammatory Totreatemphysema (breathingditficultydue todistensionoflungs) Analyticalapplication reagents(for estimation) GlucoseoxidaseandoeroxidaseGlucose Therapeuticapplications Streptokinase/urokinase Asparaginase Paoain o,-Antitrypsin Urease Cholesteroloxidase Uricase Urea Cholesterol Uricacid Lipase Triacylglycerols Luciferase Todetectbacterial contaminationoffoods Alkalinephosphatase/ Intheanalyticaltechnique lgpp-n9irleereriq?ee_qL!94 Applicationsingeneticengineering Restrictionendonucleases Genelransfer,DNAtinger pnnrng Iag DNApolymerase Polymerasechain reaction Industrialapplications Rennin Glucoseisomerase u,-Amylase Proteases Cheesepreparation Productionofhigh fructosesyrup Infoodindustryto convertstarchtoglucose Washingpowder Enzymes as therapeqtic agents 1. Streptokinasepreparedfromstreptococcus is useful for clearing the blood clots. Streptokinaseactivatesplasmaplasminogento plasminwhich,in turn,attacksfibrinto convert intosolubleproducts. Plasminogen I J Streptokinase Plasmin IFibrin-l-* Solubleproducts (clot)
  • BIOCHEMISTRY106 2. The enzyme asparaginaseis used in the treatmentof leukemias.Tumorcellsaredependent on asparagineof the host's plasma for their multiplication.By administeringasparaginase,the host'splasmalevelsof asparagineare drastically reduced.This leadsto depressionin the viability of tumor cells. Enzyrmes as analytica! reagents Some enzymes are useful in the clinical laboratoryfor the measurementof substrates, drugs,and even the activitiesof other enzymes. The biochemicalcompounds(e.g.glucose,urea, uric acid, cholesterol)can be more accurately and specifically estimated by enzymatic procedures compared to the conventional chemical methods. A good example is the estimationof plasmaglucoseby glucoseoxidase and peroxidasemethod. lmmobilized enzymes Enzymescan be used as catalyticagentsin industrial and medical applications.Some of theseenzymesare immobilizedby binding-.them to a solid, insoluble matrix which will not affect the enzyme stability or its catalytic activity. Beaded gels and cyanogen bromide activated sepharoseare commonly used for immobilizationof enzymes.The boundenzymes can be preservedfor long periodswithout lossof activity. Clucoseoxidaseand peroxidase,immobilized and coatedon a strip of paper,are used in the clinical laboratoryfor the detectionof glucosein urine. Glucose xidgg9 t Gluconicacid+ Hro, Hzoz o-Toluidine (colourless) Hzo Oxidizedtoluidine (bluecolour) The intensityof the blue colour dependson the concentrationof glucose.Hence, the strip method is usefulfor semi-quantitativeestimation of glucosein urine. Estimationof enzyme activitiesin biological fluids (particularly plasma/serum)is of great clinical importance.Enzymesin the circulation are divided into two Eroups- plasmafunctional and plasmanon-functional. l. Flasma speeific or Plastna functional enzYrnes Certainenzymesare normally presentin the plasma and they have specific functions to perform.Cenerally,theseenzyme activitiesare higher in plasmathan in the tissues.They are mostly synthesizedin the liver and enter the circulation e.g. lipoprotein lipase, plasmin, thrombin, choline esterase,ceruloplasminetc. lmpairment in liver function or genetic disordersoften leadsto a fall in the activitiesof plasma functional enzymes e.g' deficiency of ceruloplasminin Wilson'sdisease. 2, i{on-piasrma specific or plasrna non-functional enzymes These enzymes are either totally absent or oresent at a low concentration in plasma compared to their levels found in the tissues. The digestive enzymes of the gastrointestinal tract (e.g. amylase,pepsin,trypsin, lipaseetc.) present in the plasma are known as secretory enzymes. All the other plasma enzymes associatedwith metabolism of the cell are collectivefy referredto as consfitutive enzymes (e.g.lactatedehydrogenase,transaminases,acid and alkaline phosphatases,creatine phospho- kinase). Estimationof the activities of non-plasma specific enzymes is very important for the diagnosisand prognosisof severaldiseases. The normal serum level of an enzyme indicatesthe balancebetweenits synthesisand releasein the routine cell turnover.The raised enzymelevelscould be due to cellulardamage, increasedrate of cell turnover, proliferationof cells,increasedsynthesisof enzymesetc. Serum
  • Chapten 6 : ENZYMES 107 Serumenzyme (elevated) Disease(most important) Amylase Serumglutamatepyruvatetransaminase(SGPT) Serumglutamateoxaloacetaletransaminase(SGOT) Alkalinephosphatase Acidphosphatase Lactatedehydrogenase(LDH) Creatinephosphokinase(CPK) Aldolase 5'-Nucleolidase yGlutamyltranspeptidase(GGT) Aculepancreatitis Liverdiseases(hepatitis) Heartattacks(myocardialinfarction) Rickets,obstructivejaundice Cancerofprostategland Heartattacks,liverdiseases Myocardialinfarction(earlymarker) Musculardystrophy Hepatitis Alcoholism l', enzymesare convenientlyused as markersto detect the cellular damage which ultimately helps in the diagnosis of diseases. A summaryof the importantenzymesuseful for the diagnosisof specificdiseasesis given in Table6.8. Detailedinformationon the diagnostic enzymesincluding referencevalues is provided in Table 5.9. A brief account of selected diagnosticenzymesis discussed Amylase : The activity of serum amylaseis increasedin acutepancreatitis(normal 80-180 Somogyi units/dl).The peak value is observed ,ithin 8-12 hours after the onset of disease rvhich returns to normal by 3rd or 4th day. Elevatedactivityof amylaseisalsofound in urine of the patients of acute pancreatitis.Serum anrylaseis also important for the diagnosisof chronicpancreatitis,acuteparotitis(mumps)and obstructionof pancreaticduct. Alanine transaminase(ALT/SGPT): SCPT is elevated in acute hepatitis of viral or toxic origin,jaundiceand cirrhosisof liver(normal3- J,,rlUll). Aspartate transaminase (AST/SGOT) : SCOT activity in serum is increasedin myocardial iniarction and also in liver diseases(normal -1-.+itull). It may be notedthatSCPTis morespecificfor the diagnosisof liverdiseaseswhile SCOTis for heart diseases.This is mainly becauseof their cellular distribution- SCPT is a cytosomal enzyme while SCOT is found in cytosol and mitochondria. Alkalinephosphatase(ALP): lt is elevated in certainbone and liver diseases(normal3-13 KA units/dl). ALP is useful for the diagnosis of rickets, hyperparathyroidism, carcinoma of bone, and obstructive jaundice. Acid phosphatase(ACP) : lt is increased in the cancer of prostate gland (normal 0.5-4 KA units/dl).The tartaratelabileACP (normal<1 KA units/dl)is usefulfor the diagnosisand pi-ognosis of prostatecancers i.e. ACP is a good tumor marker. Lactatedehydrogenase(LDH): LDH is useful for the diagnosis of myocardial infarction, infective hepatitis, leukemia and muscular dystrophy(serumLDH normal50-200lull). LDH has five isoenzymes,the details of which are describedlater. Creatine kinase (CK) . lt is elevated in myocardial infarction (early detection) and musculardystrophy(normal10-50lUll). CK has three isoenzymes(describedlater).
  • o o E o o I m 6 n Enzymes Reference value Disease(s)in which increased l. Dig,estiveenzYmes Amylase Lipase 80-180Somogyiunits/dlor2.5-5.5pKat 0.2-1.5lu/l Aculepancreatitis,mumps(acuteparotitis),obstructioninpancreaticduct,severediabetic ketoacidosis. Acutepancreatitis,moderateelevationincarcinomaofpancreas. ll. Transaminases Alaninetransaminase(ALT)orserum glutamatepyruvatetransaminase(SGPT) Aspartatetransaminase(AST)or serumglutamateoxaloacetate transaminase(SGOT) 3-40lu/lor40-250nKat 4-45lu/lor5G-320nKat Acutehepatitis(viralor toxic),jaundice,cirrhosisof liver. Myocardialinfarction,liverdiseases,livercancer,cirrhosisot liver. lll. Phosphatases Alkalinephosphatase(ALP) (pHoptimum9-10) Acidphosphatase(ACP) (pHoptimum#S) Inadults-$13KingArmstrong(KA)units/dl or2$-90lU/lor500-1400nKat. Inchildren-l5-30Klr/dl 0.5-4KAuniWdlor2.5-12IU/' or10-100nKat.Tanaratelabile ACP-G{.9KAunits/dl Bonediseases(relatedtohigherosteoblasticactivity)-rickets,Pagets'disease,hyperpara- thyroidism,carcinomaolbone. Liverdiseases-obstructivejaundice(cholestasis),infectivehepatitis,cirrhosisofliver. Prostaticcarcinomai.e.cancerofprostategland(tartarateliabileACPseryesasa marker fordiagnosisandfollowup),Pagets'disease. lV. Enrymesof carbohydratemetabolism Aldolase lsocitratedehydrogenase(lCD) Lactatedehydrogenase(LDH) 2-6tu/l 1-4tu/l 50-200lu/lor1-5pKat Musculardystrophy,liverdiseases,myocardialinfarction,myastheniagravis,leukemias Liverdiseases(inflammatorytoxicormalignant) Myocardialinfarction,acuteintectivehepatitis,musculardystrophy,leukemia,pemicious anaemE. V. Miscellaneousenzynes Creatinekinase(GK)or creatine phosphokinase(CPK) Cholinesterase(ChEl) yGlutamyltranspeptidase(GGT) Ceruloplasmin(tenooxidase) S'-Nucleotidaseornucleotidephosphatase(NTP)2-15 lu/l 1150tu/l 2-10ru/l 5-40tu/l 2tr50mg/dl Myocardialinfarction(CKusefulforearlydetection),musculardystrophy, hypothyroidism,alcoholism. Nephroticsyndrome,myocardialinfarction Hepatitis,obstructivejaundice,tumors Alcoholism,infectivehepatitis,obstructivejaundice. Bacterialinfections,collagendiseases,cirriosis,pregnancy.
  • Chapter 6 : ENZYMES Referencevalues Disease(s)in which decreased Amylase Pseudocholinesterase(ChEll) Ceruloplasmin Glucose6-phosphatedehydrogenase(G6PD)inRBC Liverdiseases Viralhepatitis,malnutrition,livercancer, cirrhosisofliver Wilson'sdisease (hepatolenticulardegeneration) Congenitaldeficiencywithhemolyticanemia 8G-180Somogyiunits/dl 10-20tu/dl 20-50mg/dl 121260tU/1012RBC y-Glutamyl transpeptidase (GGT) : lt is a sensitivediagnosticmarkerfor the detectionof alcoholism. GGT is also increased in infective hepatitisand obstructivejaundice. Decreased plasma enzyme aetivities Sometimes, the plasma activities of the enzymesmay be lowerthan normalwhich could be due to decreased enzyme synthesis or congenital deficiency. ln Table 5.10, the decreasedplasmaenzymesin certain disorders are given. The multiple forms of an enzyme catalysing the same reaction are isoenzymesor isozymes. They, however, differ in their physical and chemicalpropertieswhich includethe structure, electrophoreticand immunological properties, K,n and V.nr" values, pH optimum, relative susceptibility to inhibitors .and degree of denaturation. Explanation for the existence of isoenzymes Many possiblereasonsare offeredto explain the presenceof isoenzymesin the livingsystems. . 1. lsoenzymes synthesized from different genese.g. malate dehydrogenaseof cytosol is differentfrom that found in mitochondria. 2. Oligomeric enzymesconsistingof more than one type of subunitse.g. lactatedehydro- genaseand creatinephosphokinase. 3. An enzyme may be activeas monomeror oligomer e.g. glutamatedehydrogenase. 4. In glycoprotein enzymes, differencesin carbohydratecontent may be responsiblefor isoenzymese.g. alkalinephosphatase. lsoenzymes of lactate dehydrogenase (LDHI Among the isoenzymes,LDH has been the most thoroughlyinvestigated. LDH whose systematicname is L-lactate- NAD+ oxidoreductase(E.C.1.'1.1.27)catalyses the interconversionof lactate and pyruvate as shown below LDfI ? cH3-cH-@oH -,----+ CH3-e-C OOH oH NAD+NADH+ H+ Lactic acld Pyruvicacld LDH has five distinct isoenzymesLDHt, LDH2, LDH3, LDHa and LDH5. They can be separatedby electrophoresis(celluloseor starch gel or agarosegel). LDHI has more positive charge and fastestin electrophoreticmobility while LDH5 is the slowest. Strdcture of LDH isoenzymes : LDH is an oligomeric(tetrameric)enzyme made up of four polypeptide subunits. Two types of subunits namely M (for muscle) and H (for heart) are producedby differentgenes.M-subunit is basic while H subunit is, acidic. The isoenzymes contain either one or both the subunitsgiving LDHI to LDH'. The characteristicfeaturesof LDH isoenzymesare given in Table5.11.
  • 110 BIOCHEMISTRY Isoenzyme Subunit constitution Principal tissueof origin Electrophoretic Whether Percentageof mobility destroyed normal serum by heat (at 60"C) in humans LDHr LDHz LDH3 HzMz HeartandRBC HeartandRBC Brainandkidney Liverandskeletalmuscle Skeletalmuscleandliver H ffi Ha HsM HMs Ma Fastest No No Partially 25To 35Yo 27ToFast LDHr LDHs Slow Slowest Yes Yes 8To 5o/o @ Significanceof differential catalytic activity : LDHl (Ha) is predominantlyfound in heart muscle and is inhibited by pyruvate- the substrate.Hence, pyruvate is not converted to lactate in cardiac muscle but is convertedto acetylCoA which enterscitric acid cycle. LDH5 (M+)is mostlypresentin skeletalmuscleand the inhibitionof thisenzymeby pyruvateis minimal, hence pyruvateis convertedto lactate.Further, LDH5haslow K. (highaffinity)while LDHl has high Km (low affinity) lor pyruvate. The differentialcatalyticactivitiesof LDHl and LDH5 in heart and skeletalmuscle, respectively,are well suitedfor the aerobic(presenceof oxygen) and anaerobic(absenceof oxygen)conditions, prevailingin thesetissues. Diagnosticimportanceof LDH : lsoenzymes of LDH have immensevalue in the diagnosisof heart and Iiver related disorders(Fi9.6.1fl. ln healthy individuals,the activity of LDH2 is higherthanthatof LDHl in serum.In the caseof myocardial infarction, LDHI is much greater than LDH2 and this happenswithin 12 to 24 hoursafter infarction.Increasedactivity of LDH< Fig. 6.15 : Electrophoresisof lactate dehydrogenase with relative proportians of isoenzymes (A) Normal serum (B) Serum from a patient of myocardial infarction (LDH, and LDH2T)(C) Serum from a patient of liver disease (LDH|T) (c)
  • ENZYMES 111 in serum is an indicatorof liver diseases.LDH activityin the RBC is 80-100 times more than that in the serum.Hencefor estimationof LDH or its isoenzymes,serumshould be totallyfree from hemolysisor elsefalsepositiveresultswill be obtained. Creatinekinase(CK)or creatinephosphokinase (CPK)catalysesthe inter-conversionof phospho- creatine(or creatinephosphate)to creatine. In healthy individuals, the isoenzyme CPK2 (MB) is almost undetectablein serum with less than 2'h of total CpK. After the myocardialinfarction(Ml), withirrthe first6_.1g hours,CPK2increasesin the serumto as hieh as 209lo(against2oh normal).CpK) isoenzvnre,s not elevated in skeletal muicle disorders. Therefore,estimationof the enzyme CpKz (MB) is the earliest reliable indication of mvoiardial infarctian. As many as six isoenzymesof alkaline phosphatase(ALP)have been identified.ALp is a monomer, the isoenzymes are due to the difference in the carbohydrate content (sialic acid residues).The most importanr ALP isoenzymesare cx1-ALp, u2-heat labile ALP, o,2-heatstable ALp, pre-B ALp, y-ALp etc. Increase in cr2-heatlabile ALp suggests hepatitiswhereas pre p-ALp indicates ltone diseases. subunits-M (muscle)or B Isoenzyme Subunit Tissueof origin cPKl cPK2 cPK3 BB MB MM Brain Heart Skeletalmuscle BIOMEDICAL/ CHHIGALCSNCEPTS t:;t In the liuing system, the regulation oJ enzgme qctiuities occurs through allosteric inhibition, actiuation of lotent enzymes, compartmentation of metabot-icpathways, control of enzyme synthesis and degrodation. w Feedback(or end product) inhibition is a specializedform oJ allosteric inhibition that controls seuerol metabolic pathways e.g. CTP inhibits aspartote transcorbamoylase; cholesterol inhibits HMG coA reductase. The end priduct inhibition is ufmost important to cellulareconomysincea compound is synthesizedonlg when required. r"-; Certain RNA molecules(ribozymes)function as non-proteinenzymes.It is belieuedthat ribozgmeswere lunctioning as biocatalystsbefore the orrurr"r4 of protein enzymes during euolution. r'>'Certain enzymesare utilized os therapeutic agents. Streptokinosein usedto dissolue blood clots in circulation while asparaginoseis emplogedin the treatmentof leukemias. r':' Determination of serum enzyme actiuitiesis of great importance t'or the diagnosisof seueraldiseoses(refer Table 6.8). rt'' Lowered body temperature (hypothermia) is accompained by o decrease in enzyme actiuities' This principle is exploited to reduce metobolic demand. during open heart Phosphocreatin"+ -9{--------lCreatine CPK exists isoenzyme rs ADP as three a dimer ATP isoenzymes. Each composed of two (brain)or both. surgery or transportotion of organslor transplantationsurgery.
  • lt2 BIOCHEMISTF|Y i o E N ul 0 6 12f8243036 424A*6066724 5 6 7 I 9 10 11 Hours Days Creatine phosphokinase (precisefy isoenzyme MB) is the first enzyme to be releasedinto circulationwithin 6-18 hoursafterthe infarction. Therefore,CPKestimationis highly usefulfor the early diagnosisof Ml. This enzyme reachesa peak value within 24-30 hours, and returnsto normal level by the 2nd or 3rd day. Aspartate transaminase(ASTor SCOT) rises sharplyafterCPK,and reachesa peakwithin 48 hours of the myocardial infarction.AST takes 4-5 days to returnto normal level. Lactatedehydrogenase(LDHl) generallyrises from the second day after infarction, attains a peak by the 3rd or 4th day and takes about 10-15 daysto reachnormal level.Thus,LDH is the fast enzymeto rise and also the last enzyme to returnto normal level in Ml. Cardiac troponins (CT) : Although not enzymes/ the proteins cardiac troponins are highly useful for the early diagnosisof Ml. Among these, troponin I (inhibitory element of actomysin ATPase)and troponin f (fropomysin bindingelement)are important.Cardiactroponin | (CTl) is releasedinto circulation within four hoursafterthe onsetof Ml, reachesa peak value by 12-24 hours,and remainselevatedfor about a week. The protein myoglobin is also an early marker for the diagnosisof Ml. Myoglobin is however, not commonly used as it is not specific to cardiac diseases.ln the Table 6.12, a summary of the diagnosticmarkersused in Ml is given. Hnzymes in liver diseases The following enzymes-when elevated in serum-are useful for the diagnosis of liver dysfunction due to viral hepatitis (jaundice), toxic hepatitis,cirrhosisand hepaticnecrosis 1. Alaninetransaminase; 2. Aspartatetransaminase; 3. Lactatedehydrogenase; The enzymes that markedly increase in intrahepaticand extrahepaticcholestasisare : '1. Alkaline phosphatase, 2. 5'-Nucleotidase Fig. 6.16 : Enzyme paftern in myocadial infarction (CPK-Creatine phosphokinase; SGOT-Serum Fo;t;nritar}iiries sf alcohof, r$e",fraydrogenr.*se Alcohol dehydrogenase (ADH) has two heterodimer isoenzymes. Among the white Americans and Europeans,cx,p1isoenzyme is predominantwhereasin Japaneseand Chinese (Orientals)oB2is mostlypresent.The isomerop2 more rapidly convertsalcohol to acetaldehyde. Accumulation of acetaldehvdeis associated with tachycardia (increasein heart rate) and facial flushing among Orientals which is not commonlv seen in whites. lt is believed that Japaneseand Chinesehave increasedsensitivity to alcohol due to the presenceof ap2-isoenzyme of ADH. For the right diagnosisof a particulardisease, it is always better to estimate a few (three or more) serum enzymes, instead of a single enzyme. Examples of enzyme patterns in importantdiseasesare given here. Hnrymes in nnyoeardial infarction Theenzymes- namelycreatinephosphokinase (CPK),aspartatetransaminase(AST)and lactate dehydrogenase(LDH)-are important for the diagnosis of myocardial infarction (Ml). The elevationof theseenzvmesin serumin relationto hours/daysof Ml is given in the Fig.6.l6.
  • Chapter 6 : ENZYMES 113 Diagnosticmarker Timeof peak elevation Timeof return to normal level Diagnostic importance 4-Ohrs 20-25hrs Earliestmarker,howevernotcardiacspecific. CardiactroooninI 12-24hrs 5-9days Earlymarkerandcardiacspecific. CardiactroponinT 18-36hrs 5-14days Relativelyearlymarkerandcardiacspecific. However,elevatedinotherdegenerativediseases. Creatinephosphokinase(MB) 20-30hrs 24-48hrs Cardiacspecificandearlymarker. Laclatedehydrogenase(LDHl) 48-72hrs 10-15days Relativelylatemarkerandcardiacspecific. Asparlatetransaminase 3048hrs 4-6days Notcardiacspecific, Serum-y-glutamyltranspeptidaseis useful in the diagnosisof alcoholic liver diseases. Enzymes in muscle diseases Inthemusculardystrophies,probablydueto the increased leakage of enzymes from the damagedcells, serum levels of certain muscle enzymesare increased.These include creatine phosphokinase, aldolase and aspartate transaminase.Of these,CPK is the most reliable indicator of muscular diseases,followed by aldolase. Enzymes in cancers Increase in the serum acid phosphatase (tartaratelabile) is specificfor the detectionof prostaticcarcinoma. lNote : Prostate specific antigen (PSA; mol wL 32 KD), though not an enzyme, is a more reliable marker for the detection of prostate cancer. Normal serum concentrationof PSA is 1-4 n{mll. A non-specificincreasein certain enzymes like LDH, alkalinephosphataseandtransaminase may be associatedwith malignancyin any part of the body. p-Clucuronidaseestimationin urine is useful for detecting the cancers of urinary bladder, pancreasetc.
  • 114 BIOCHEMISTFIY r' Enzymesare the protein biocatarystssynthesizedby the 'iuing cers. '!ii,iir r!ri'"r c/osse's---'oxidoreductaies,transferis"r, nvirZnu,r, They are classified tyases, isomerases 2' An enzgm" o to::,r!r-:in its action,possessingactiuesite, where the substrate bindstoform enzgme-substrqtecomplex, i"f;;. the productis t'ormed. 3. Factorslike cont tr;ti,,:";":i",:!riii:{ii%Ti;'i!"Ji,i;:::{:;:::::;:i,xtJ,.!J{j,i{T,zT n ,1,i":,I'f*f."iX'J'"i,,J,i2,,:"Y:::!,bvreuersibte(competitiue,and non-competitiue), S Many enzymesrequire .the prerenceot' non-protein substancescalled cofactors(coenzyntes)for 'i;;:::7" Most of tt''' i*r"v-o-Lre"iriwtiues of B-comptex-uitamins(e.g. NAD+, FAD, 6 The mechanism "I,:rrr",a.ctjo2 is e.xproinedbv tock and keymoder(oJFischer),marerecenttyinducedfit madet(of Koshtand),"i ,"triiiJililn ,n.orr. ' [l"o;:':ff:r7:;;::,'n" rateof reactionthroughacid-basecatatysis,couarentcatarysis t ,;,!l;i;:,if"i1n]lf,*, thereisa constantregutationof enzymeteuersbroughtaboutbv etc. sm,actiuationot'proenzgmes,synthesisanddegrad"ri"i.t "*i^Z 9. Estimation of sert serumo*,u;*-;;,:fi,;:::T::,:,,'J",::;i:!:':,,,;"!,:*i;i,f:::;:,?1.'f:",:1j,,:::::;hepatitis; aspartate,. trarraminas., lorror" dihgdrogenase"(tDH) and creatinephosphokinase(CpK) ^ ^uoroi,"ot i,i.rorrrror,, alkalinephosphatasein rickets and i,n:::i::i::;::;:f o,,iainolin'io''.-"'nprostatic,o,,,no-[; vstutamv!transpep- L0' Isoenzymesare the,,murtip,refotrysof an enzyme catarysingthe same reoctian whichhoweue4dift'er in their phvsicai'rri ii"^,car propertie".iD; hasfive isoenzgmes i:i::ri:: hasthree'roui""a ciiz;;;'r",u importantin thedragnosisot'myocardiar
  • Chapter 6 : ENZYMES 115 I. Essayquestiosrs 1. What areenzymes?Describetheir classificationand nomenclature. 2. Write an accountof the variousfactorsaffectingenzymeactiviti. 3. Describethe mechanismof enzymeaction. 4. What arecoenzymes?Write brieflyon the role of coenzymesin enzymeaction. 5. Writean accountof the importanceof serumenzymesin the diagnosisof diseases. lL Short notes (a) Enzymespecificity,(b) Competitiveinhibition, (c) Coenzymes,(d) Allostericenzymes/ (e)lsoenzymes,(f)K, value,(g)Serumenzymesin myocardialinfarction,(h)Lactatedehydrogenase, (i) Roleof metalsin enzymeaction,(j) Activesite. lIL Fill in the blanks 1. The literalmeaningof enzymeis - 2. The classof enzymesinvolvedin syntheticreactionsare 3. The non-proteinpartof holoenzyme 4. Enzymeslosethe catalyticactivityat temperatureabove 70oCdue to 5. Examplesof two enzymescontainingzinc are and 6. The placeat which substratebindswith the enzyme 7. Theenzymeglucose6-phosphatedehydrogenaserequiresthe coenzyme 8. The E.C.numberfor alcoholdehydrogenasers 9. Phsophofructokinaseis allostericallyactivatedby 10. The very firstenzymeelevatedin serumin myocardialinfarction IV. Multiple choice questions 11. Pepsinis an examplefor the classof enzymesnamely (a) Oxidoreductases(b) Transferases(c) Hydrolases(d) LiSases. 12. The coenzymenot involvedin hydrogentransfer (a)FMN (b) FAD(c) NADP+(d) FH4. 13. In the feedbackregulation,the end productbindsat (a)Activesite(b)Allostericsite (c) E-Scomplex(d) Noneof these. 14. y-Clutamyltranspeptidaseactivityin serumis elevatedin (a)Pancreatitis(b)Musculardystrophy(c) Myocardialinfarction(d)Alcoholism. 15. In recentyears/a non-proteincompoundhas been identifiedto bring about catalysisin biologicalsystem.The nameof the compoundis (a)DNA (b) RNA (c) Lipids(d)Carbohydrates.
  • Vitamins t, ++ Fat Water soluble soluble I t is difficult to define vitamins precisely. I vitamins may be regarded as organic compounds required in the diet in small amounts to perform specific biological functions for normal maintenance of optimum growth and health of the organism. The bacterium E.coli doesnot requireanyvitamin,as it can synthesize all of them. lt is believedthat during the course of evolution,the ability to synthesizevitamins was lost. Hence, the higher organismshave to obtainthemfrom diet.Thevitaminsarerequired in small amounts,since their degradationis relativelyslow. History and nomenclature In the beginning of 20th century, it was clearly understoodthat the diets containing purified carbohydrate,protein,fat and minerals were not adequateto maintainthe growth and health of experimentalrats, which the natural foods (suchas milk) could do. Hopkins coined the term accessoryfactorsto the unknown and essentialnutrientspresentin the naturalfoods.Funk(1913)isolatedan active principle (an amine) from rice polishingsand, later in veast,which could cure beri-beri in pigeons. He coined the term vitamine (Creek : vita-life)to the accessoryfactorswith a belief thatall of them were amines.lt was laterrealised that only few of them are amines. The term vitamin,however,is continuedwithout the final letter 'e'. The usageof A, B and C to vitamins was introducedin 1915 by McCollum and Davis. They first felt there were only two vitamins- fat soluble A and water soluble I (anti-beriberi factor).Soon another water soluble anti-scurvy factor namedvitamin C was described.Vitamin A was later found to possesstwo components- one that preventsnightblindness(vitaminA) and anotheranti-ricketfactornamedasvitamin D. A fat solublefactorcalledvitaminE,in theabsence of which ratsfailed to reproduceproperly,was discovered. Yet another fat soluble vitamin concernedwith coagulationwas discoveredin mid 1930s.lt was named as vitamin K. In the
  • Chapter 7 : VITAMINS 777 sequenceof alphabetsit should have been F, but K was preferred to reflect its function (koagulation). As regardsthe water solublefactors,vitamin C was identifiedasa pure substanceand named as ascorbicacid. Vitamin B was found to be a complexmixtureand nomenclaturealsobecame complex. B1 was.clearlyidentifiedas anti-beri- beri factor. Many investigatorscarried out intensiveresearchbetween192Oand 1930 and went on naming them as the water soluble vitamins82, 83, 84,85,86, 87, 86, Bg,819,811 and 812. Some of them were found to be mixturesof alreadyknown vitamins.And for this reason,a few members (numbers!)of the B- complex series disappearedfrom the scene. Exceptfor 81, Bz, Bo and 812,namesare more commonly usedfor other B-complexvitamins. Glassification of vitamins There are about 15 vitamins, essentialfor humans.They are classifiedas fat soluble (A, D, E and K) and water soluble (C and B-group) vitamins as shown in the Table 7.1. The B-complexvitamins may be sub-dividedinto energy-releasing(81, 82, 86, biotin etc.) and hematopoietic (folic acid and 812).Most of the water soluble vitamins exert the functions through their respectivecoenzymeswhile only one fat solublevitamin(K)hasbeen identifiedto function as a coenzyme. $ynthesis of vitannims by intestina! bacteria Vitamins, as per the definition, are not synthesizedin the body. However,the bacteria of the gut can produce some of the vitamins, required by man and animals. The bacteria mainly live and synthesizevitaminsin the colon region,where the absorptionis relativelypoor. Some of the animals (e.g. rat, deer etc.) eat their own feces, a phenomenon known as coprophagy. As far as humansareconcerned,it is believed that the normal intestinalbacterialsyntfiesigand absorption of vitamin K and biotin may be sufficient to meet the body requirements. For other B-complex vitamins, the synthesisand absorptionare relativelyless.Administrationof anitibioticsoften kills the vitamin synthesizing bacteria present in the gut, hence additional consumptionof vitaminsis recommended. VitaminA VitaminD I V1aminE VitaminC VitaminK I l-Folic acid(Bn) L-Vitamin8', (cyanocobalamin) --.._
  • 118 BIOCHEMISTRY Fa{ soluble vitamins-general The four vitamins, namely vitamin A, D, E, and K are known as fat or lipid soluble.Their availabilityin the die! absorptionand transport are associatedwith fat. Thev are soluble in fats and oils and also the fat solvents (alcohol, acetoneetc.).Fatsolublevitaminscan be stored in liver and adiposetissue.They are not readily excretedin urine. Excessconsumptionof these vitamins (particularlyA and D) leads to their accumulationand toxic effects. ' Alf the fat soluble vitamins are isoprenoid compounds, since they are made up of one or more of five carbon units namely isopreneunits (-CH=C.CH3-CH=CH-). Farsolublevitamins perform diverse functions. Vitamin K has a specificcoenzymefunction. Water sCIluble vitamlels*seneral The water soluble vitamins are a heterogenousgroup of compounds since they differ chemically from each other. The only common character shared by them is their solubility in water. Most of these vitamins are readilyexcretedin urine and they are not toxic to the body. Water soluble vitamins are not stored in the body in large quantities(except 812).For this reason,they must be continuously supplied in the diet. Generally, vitamin deficiencies are multiple rather than individual with overlapping symptoms. lt is often difficult to pinpoint the exact biochemical basis for the symptoms. The water soluble vitaminsform coenzymes (Refer Table 5.3) that participate in a variety of biochemical reactions,relatedto either energy generationor hematopoiesis.lt may be due to this reasonthat the deficiencyof vitaminsresults in a number of overlapping symptoms. The common symptomsof the deficiencyof one or more vitamins involved in energy metabolism include dermatitis,glossitis(red and swollen tongue),cheilitis(ruptureat the cornersof lips), diarrhea, mental confusion, depressionand malaise. Deficiencyof vitamins81, 86 and B12is more closelyassociatedwith neurologicalmanifestations. Vitamers The term vitamers representsthe chemically similar substancesthat possess qualitatively similar vitamin activity.Some good examplesof vitamersare given below . Retinol,retinaland retinoicacid are vitamers of vitamin A. . Pyridoxine,pyridoxal and pyridoxamineare vitamersof vitamin B.. In the following pages, the individual membersof the fat soluble and water soluble vitamins are discussed with regard to the chemistry,biochemicalfunctions,recommended dietary/dailyallowances(RDA),dietarysources, deficiencymanifestationsetc. The fat solublevitamin A, as such is present only in foods of animal origin. However, its provitamins carotenes are found in plants. It is recordedin the historythat Hippocrates (about 500 B.C.) cured night blindness.He prescribedto the patientsox liver (in honey), which is now known to contain high quantityof vitaminA. Chennistry In the recent years, the term vitamin A is collectivelyused to representmany structurally related and biologically active molecules (Fig.7.1).The term retinoids is often used to include the natural and synthetic forms of vitamin A. Retinol, retinal and retinoic acirl arc regardedas vitamersof vitamin A. 1. Retinol(vitaminA alcohol): lt is a primary alcoholcontainingp-iononering. Thesidechain has two isoprenoid units,four double bonds and one hydroxylgroup.Retinolis presentin animal tissuesasretinylesterwith longchainfattyacids.
  • Ghapter 7 : VITAMINS 119 -C=O I OH Retinal -C=O I H Retinolc acid p-lonone Fig. 7.1: Structuresof vitaminA andrelatedcompounds(Redcolourreptesents thesubstituentgroupsin therespectivecompounds). 2. Retinal(vitamin A aldehyde): This is an aldehyde form obtained by the oxidation of retinol. Retinaland retinol are interconvertible. Previously,the name retinine was used for retinal. 3. Retinoic acid (vitamin A acid) : This is producedby the oxidationof retinal.However, retinoicacid cannotgive riseto the formationof retinalor retinol. 4. p-Carotene(provitamin A) : This is found in plant foods. lt is cleaved in the intestineto produce two moles of retinal. ln humans,this conversion is inefficient, hence p-carotene possessesabout one-sixth vitamin A activity comparedto that of retinol. Absorption, transport and mobilization Dietary retinyl esters are hydrolysed by pancreaticor intestinalbrush border hydrolases in the intestine,releasingretinol and free fatty acids. Carotenesare hydrolysed by p-carotene l5-1S'-dioxygenaseof intestinal cells to release 2 molesof retinalwhich is reducedto retinol.In As and when needed,vitamin A is released from the liver as free retinol. lt is believedthat zinc plays an important role in retinol mobilization. Retinol is transported in the circulation by the plasma retinol binding protein (RBP; mol. wt. 21,000) in associationwith pre-albumin.The retinol-RBPcomplex binds to specific receptors on the cell membrane of peripheral tissue and enters the cells. Many cells of target tissuescontain a cellular retinol- binding protein that carries retinol to the nucleus and binds to the chromatin (DNA). It is here that retinol exerts its function in a manner analogous to that of a steroid hormone. BIOCHEMICALFUNCTIONS Vitamin A is necessaryfor a variety of functions such as vision, proper growth and differentiatioryreprbductionand maintenanceof epithelial cells. In recent years, each form of vitamin A has been assignedspecificfunctions (Fig.7.3). Vitamin A and vision : The biochemicalfunc- tion of vitamin A in the processof vision was first elucidated by Ceorge Wald (Nobel Prize 1968). The events occur in a cyclic process known as Rhodopsin cycle or Wald's visual cycle (Fig.7.4).
  • BIOCHEMISTFIY120 lntestinalcell p-Carotene j Retinal J Retina All-fransretinol II + All-transretinal II J Visualcycle (SeeFig.7.a) Nuclear receptor i j v Specificproteins I I + Celldifierentiation Chylomicrons RBP-t Retinol-RBP Fig.7.2: summaryof vitaminA absorption,transportandbiochemicalfunctions - (FFA-Freefaw acid;RBP-Retinolbindingprotein)'
  • Chapter 7 : VITAMINS 121 Retinal RetinylPhosphate (visualcycle) (glycoproteinsynthesis; I+ Retinoicacid (steroidhormone-growthand differentiation) B-Carotene (antioxidant) I+ Retinol (steroidhormone--{roMh and difterentiation) Dark adaptationtime : When a personshifts from a brightlightto a dim light(e.9.entry into a dim cine theatre), rhodopsin stores are depleted and vision is impaired. However, within a few minutes,known as dark adaptation time, rhodopsin is resynthesizedand vision is improved.Dark adaptationtime is increasedin vitamin A deficient individuals. Bleachingof rhodopsin: When exposedto light, the colour of rhodopsinchangesfrom red to yellow, by a processknown as bleaching. Bleachingoccurs in a few millisecondsand many unstableintermediatesare formed during this orocess. Rhodopsin Prelumirhodopsin-----) Lumirhodopsin All-trans-retinal+Opsin+- Metarhodopsinll+--MetartdopsinI Visualcascadeand cGMP : When lightstrikes the retina,a number of biochemicalchanges leadingto membranehyperpolarizationoccur resultingin the genesisof nerve impulse.The hyperpolarizationof the membrane is brought aboutby a visualcascadeinvolvingcyclicCMP. When a photon (from ligh$ is absorbed by rhodopsin, metarhodopsinll is produced. The protein transducin is activatedby metarhodopsin ll. Thisinvolvesan exchangeof CTPfor CDP on inactive transducin. The activated transducin activatescyclic GMP phosphodiesterase.This Light (photon) Nerve impulse 11-cls-retinal('--"'-.--- | lsomerase I -------------+ All-i,'ansretinal Flg.7.3: Summaryof thefunctions of vitaminA compounds. Rods and cones The retina of the eye possessestwo types of cells-rods and cones.The humaneve hasabout 10 million rods and 5 million cones.The rods are in the peripherywhile conesareat the centre of retina. Rods are involved in dim light vision whereascones are responsiblefor bright light and colour vision.Animals-such as owls and catsfor which night vision is more important- possessmostly rods. Wald's visual cycle Rhodopsin(mol. wt. 35,000) is a conjugated proteinpresentin rods.lt contains11-crsretinaland the proteinopsin.Thealdehydegroup(of retinal)is linkedto e-aminogroupof lysine(ofopsin). The primary event in visual cycle, on exposureto light, is the isomerizationof 11-cis: retinal to all-trans retinal. This leads to a conformational change in opsin which is responsiblefor the generationof nerve impulse. The all-trans-retinalis immediately isomerized by retinal isomerase(of retinal epithelium).to 11-cis-retinal.This combines with opsin.:to regeneraterhodopsin and complete the visual cycle (Fig,7.4).However,the conversionof all frans-retinalto 11-crs retinal is incomplete. Therefore, most of the all-frans-retinal is transportedto the liverand convertedto all-frans retinolby alcbhol dehydrogenase.The all-trans- retinol undergoesisomerizationto 11-crsretinol which is then oxidized to 11-cis retinal to participatein the visualcycle. 11-cts-retinol+- l1ferf+All-trane.retinol Flg.7.4: Wald'sulsualcycle.
  • 122 BIOCHEMISTRY Fig.7.5 : The visualcascadeinvolvingcyclicguanosine monophosphate(3"5'-cGMP). enzyme degradescyclic CMP ir;he rod cells (Fi9.7.5).A rapid decreasein cyclic GMP closes the Na+ channelsin the membranesof the rod cells.This resultsin hyperpolarizationwhich is an excitatoryresponsetransmittedthrough the neuronnetworkto the visualcortexof the brain. Golour vision Cones are specializedin bright and colour vision. Visual cycle comparableto that present in rods is alsoseenin cbnes.The colour vision is governed by colour sensitive pigments- porphyropsin (red), iodopsin (green) and cyanopsin (hlue). All thesepigmentsare retinal- opsin complexes.When bright light strikesthe retina, one or more of these pigments are bleached,dependingon the particularcolourof light. The pigmentsdissociateto all-trans-retinal and opsin,as in the caseof rhodopsin.And this reactionpasseson a nerveimpulseto brain as a specific colour-red when porphyropsinsplits, greenwhen iodopsinsplitsor bluefor cyanopsin. Splitting of these three pigments in different proportionsresultsin the perceptionof different coloursby the brain. Other biochemical functions of vitamin A '1. Retinol and retinoic acid function almost like steroid hormones.They regulatethe protein synthesisandthusare involvedin the cell growth and differentiation. 2. VitaminA is essentialto maintainhealthv epithelialtissue.This is due to the fact that retinoland retinoicacid are requiredto prevent keratinsynthesis(responsiblefor horny surface). 3. Retinylphosphatesynthesizedfrom retinol is necessary for the synthesis of certain glycoproteinq which are requiredfor growth and mucus secretion. 4. Retinoland retinoicacid are involvedin the synthesisof transferrin,the iron transport protein. 5. VitaminA is consideredto be essentialfor the maintenanceof proper immune systemto fight againstvariousinfections. 6. Cholesterolsynthesisrequiresvitamin A. Mevalonate,an intermediatein the cholesterol biosynthesis,is diverted for the synthesisof coenzyme Q in vitamin A deficiency. lt is pertinentto notethat the discoveryof coenzyme Q was originallymade in vitamin A deficient animals. 7. Carotenor'ds(most important p-carotene) function as antioxidantsand reduce the risk of cancers initiated by free radicals and strong oxidants.p-Caroteneis found to be beneficialto prevent heart attacks.This is also attributedto the antioxidantproperty. Recommended dietary allowance (RDA) The daily requirement of vitamin A is expressedas retinol equivalents(RE)ratherthan InternationalUnits (lU). 1 retinol equivalent=1 lrg retinol =6 Pg P-carotene =12 pg othercarotenoids =3.33 lU of vitaminA activityfrom retinol ='10 lU of vitamin A activityfrom p-carotene The RDA of vitamin A for adults is arouno !"Q0Oretiytolequivalents(3,500lU) for man and 800 retinol equivalents(2,500 lU) for woman. Rhodopsin lu'no'o" Metarhodopsinll Phosphodiesterase (inactive) 3',5'-cGMP 51GMP
  • Chapter 7 : VITAMINS 123 One InternationalUnit (lU) equalsto 0.3 mg of retinol.The requirementincreasesin growing children,pregnantwomen and lactatingmothers. Dietary sources Animal sourcescontain (preformed)vitamin A. The bestsourcesare liver, kidney,egg yolk, milk, cheese,butter.Fish(codor shark)liveroils are very rich in vitaminA. Vegetable sources contain the provitamin A-carotenes.Yellow and dark green vegetables and fruits are good sourcesof carotenese.g. carrots,spinach,amaranthus,pumpkins,mango, papaya etc. Vitamin A deficiency Thedeficiencysymptomsof vitaminA are not immediate,sincethe hepaticstorescan meetthe body requirementsfor quite sometime (2-4 months). The deficiency manifestationsare relatedto the eyes,skin and growth. Deficiency manifestationsof the eyes: Nrghf blindness(nyctalopia) is one of the earliest symptoms of vitamin A deficiency. The individualshave difficultyto see in dim light since the dark adaptation time is increased. Prolonged deficiency irreversibly damages a numberof visualcells. Severe deficiency of vitamin A leads to xerophthalmia. This is characterizedby dryness in conjunctivaand cornea,and keratinizationof epithelialcells. In certain areasof conjunctiva, white triangularplaquesknown as Bitot's spots are seen. lf xerophthalmiapersisitsfor a long time, cornealulcerationand degenerationoccur. This resultsin the destructionof cornea,a condition referred to as keratomalacia, causing total blindness.VitaminA deficiencyblindnessismostly commonin childrenof the developingcountries. Other deficiency manifestations Effect on growth : Vitamin A deficiency resultsin growth retardationdue to impairment in skeletalformation. Effect on reproduction : The reproA)tiu" system is adversely affected in vitamin A deficiency.Degenerationof germinalepithelium leadsto sterilityin males. Effect on skin and epithelial cells : The skin becomes rough and dry. Keratinization of epithelial cells of gastrointestinaltract, urinary tract and respiratorytract is noticed.This leadsto increasedbacterialinfection.VitaminA deficiencv is associatedwith formationof urinarystones. The plasmalevel of retinol binding proteinis decreasedin vitamin A deficiency. Hypervitaminosis A Excessiveconsumptionof vitamin A leadsto toxicity. The symptomsof hypervitaminosisA include dermatitis(dryingand rednessof skin), enlargementof liver, skeletal decalcification, tenderness of long bones, loss of weight, irritability,lossof hair, joint painsetc. Total serum vitamin A level (normal 20-50 pgldl) is elevated in hypervitaminosisA. Free retinolor retinolbound to plasmalipoproteinsis actuallyharmfulto the body. lt is now believed that the vitamin A toxicosissymptomsappear only after retinol binding capacity of retinol binding proteinexceeds. Higher concentrationof retinol increasesthe , synthesisof lysosomalhydrolases.The manifes- tations of hypervitaminosisA are attributedto the destructiveaction of hydrolases,particularly on the cell membranes. Beneficial effects of 0.carotene Increased consumption of p-carotene is associatedwith decreasedincidence of heart attacks,skin and lung cancers.This is attributed to the antioxidantrole of p-carotenewhich is independentof its role as a precursorof vitamin A. Ingestionof high dosesof p-carotenefor long periodsare not toxic like vitamin A. Vitamin D is a fat soluble vitamin. lt resemblessterolsin structureand functionslike a hormone.
  • 124 BIOCHEMISTRY Ftg.7.6 : Formationol ergocalciferolfromergosterol. The symptomsof rickets and the benefical effectsof sunlight to prevent rickets have been known for centuries.Hess(1924) reportedthat irradiationwith ultravioletlight induced anti- rachitic activity in some foods. Vitamin D was isolatedby Angus(1931)who namedit calciferol. Ghemistry Ergocalciferol(vitamin D2) is formed from ergosteroland is present in plants (Fi9.7.6). Cholecalciferol(vitaminD3)is found in animals. Both the sterolsare similar in structureexcept that ergocalciferol has an additional methyl group and a double bond. Ergocalciferol and cholecalciferolare sourcesfor vitamin D activitv and are referred to as provitamins. During the courseof cholesterolbiosynthesis (Chapter l4), 7-dehydrocholesterol is formed as an intermediate.On exposureto sunlight, 7-dehydrocholesterolis converted to chole- calciferol in the skin (dermis and epidermis) (Fig.2.V Vitamin D is regarded as sun-shine vitamin. The synthesisof vitamin D3 in the skin is proportionalto the exposureto sunlight. Dark skin pigmentlm-qianjn)-adversly influences the synthesis'ofcholecalciferol. Absorption, tlansport and storage Vitamin D is absorbedin the small intestine for which bile is essential.Through lymph, vitaminD entersthe circulationboundto plasma a2-globulinand is distributedthroughoutthe bodv. Liverand othertissuesstoresmallamounts of vitamin D. METABOLISM AND BIOCHEMICAL FUNCTIONS Vitamins D2 and D3, as such, are not biologically active. They are metabolized identicallyin the body and conveftedto active forms of vitamin D. The metabolism and biochemicalfunctionsof vitaminD aredepicted in Fig.7.8. Synthesis of 1,25-DHCC Cholecalciferolis first hydroxylatedat 25th position to 25-hydroxycholecalciferol(25-OH o3) bv a specific hydroxylasepresentin liver. 25-OH D3 is the major storageand circulatory form of vitamin D. Kidney possessesa specific enzyme, 25-hydroxycholecalciferol (calciol) l -hydroxylase which hydroxylates 25-hydroxy- cholecalciferolat position 1 to produce 1,25- dihydroxycholecalciferol (1,2|-DHCC). 1,25 DHCC contains3 hydroxylgroups(1,3 and 25 carbon) hence referredto as calcitriol. Both the hydroxylase enzymes (of liver and kidney) requirecytochromePa56,NADPH and molecular oxygen for the hydroxylation process. The synthesisof calcitriol is depicted in Figs.7,7 and 7.8. Regulation of the synthesis of 1125.-DHCC The concentrationof 1,25-DHCCis regulated by plasma levels of calcium and phosphate. They control hydroxylationreactionat position 1. Low plasmaphosphateincreasesthe activity of 25-hydroxycholecalciferol1-hydroxylase.Low plasma calcium enhancesthe production of parathyroid hormone which in turn activates 1-hydroxylase.Thus the action of phosphateis directwhile thatof calciumis indirecton kidnev 1-hydroxylase. Ergocalclferol(Dr)
  • Ghapter 7 : VITAMINS 125 7-Ilehydrocholestercl (animals) Biochemical functions Calcitriol(1,25-DHCC)is the biologically active(ormof vitaminD.lt tegulatesthe plasma Ievelsof calciumand phospha,fe.Calcitriolacts at 3 differentlevels(intestine,kidneyandbonel to maintainplasmacalcium(normal9-11mg/dl). 1. Action of calcitriol on the intestine: Calcitriof increlsesthe intestinalabsorptionof calciumand phosphate.In the intestinalcells, calcitriolbindswith a cytosolicreceptorto form a calcitriol-receptorcomplex.Thiscomplexthen approachesthe nucleusand interactswith a specificDNA leadingto the synthesisof a specificcalcium binding protein.This protein increasesthe calciumuptakeby the intestine. The mechanismof actionof calcitriolon the targettissue(intestine)issimilarto theactionof a steroidhormone. 2. Action of calcitriolon the bone : In the osteoblastsof bone,calcitriolstimulatescalcium uptakefor depositionascalciumphosphate,Thus calcitriolisessentialforboneformation.Thebone isanimportantreservoirofcalciumandphosphate. Calcitriol along with parathyroidhormone increasesthe mobilizationof calcium and phosphatefromthebone.Thiscauseselevationin theplasnlacalciumandphosphatelevels. 3. Action of calcitriol on the kidney : Calcitriolis also involvedin minimizingthe excretionof calciumandphosphatethroughthe kidney, by decreasingtheir excretion'and enhancingreabsorption. The sequenceof eventsthat take place in responseto low plasmacalciumconcentration andthe actionof calcitriolon intestine,kidney and bone,ultimatelyleadingto the increasein plasmacalcium is given in Fi9.7.9. 24,25-Dihydroxycholecalciferol(24,25-DHCC is anothermetaboliteof vitaminD, lt is also synthesizedin thekidneyby 24-hydroxylase.The exactfunctionof 24,2S-DHCCisnotknown.lt is believedthat when'calcitriolconcentrationis adequate,24-hydroxylaseacts leadingto the synthesisof a less importantcompound24, 25-DHCC. In this way, to maintain the homeostasisof calcium,synthesisof 24,25-DHCC is alsoimportant. Sunlight 4Skin 25-Hydroxycholecalciferol (Calcidiol) II Calcidiol1o-hydroxylase | (kidney) J 1,25-Dlhydrorycholecalclferol (1,25DHCCor calcitrlol) 4
  • BIOCHEMISTFIY Skin 7-Dehydrocholesterol !*ro*-t*a* Boneformation andturnover lntestine calcitriol(r) JRecentor(J) Calcitriol ffi8lrr Calciumbinding protein Plasma Ca Ca2*absorption
  • Chapter 7 : VITAMINS 127 Fig. 7.9 : Summary of the action of calcitiol in elevating plasma calcium. Yitamin D is a hormone and not a vitamin-justification Calcitriol (1,25-DHCC)is now considered - an important calciotropic hormone,. while :.olecalciferolisthe prohormone.Thefollowing :"aracteristicfeaturesof vitamin D (comparable .r tn hormone)justify its statusas a hormone. I . Vitamin D3 (cholecalciferol)is synthesized in the skinby ultra-violetraysof sunlight. l. The biologicallyactiveform of vitaminD, :a citriol is producedin the kidney. 3. Calcitriol has target organs-intestine, lo.e and kidney,where it specificallyacts. i. Calcitriol action is similar to steroid lnnnones. lt binds to a receptor in the cytosol :-"0 ihe complex acts on DNA to stimulatethe ,,-:hesis of calcium bindingprotein. J. {ctinomycin D inhibits the action of :: :ri:'iol. This supportsthe view that calcitriol :rets its etfecton DNA leadingto the synthesis :- R.{ (transcription). i Calcitriol synthesisis self-regulatedby a -:e:3ack mechanismi.e.,calcitrioldecreasesits r"".- Snthesis. Recommended dietary allowance {RDAI --t iaill' requirementof vitamin D is 400 Wenntknal Units or 10 mg of cholecalciferol. In countries with good sunlight (like the RDA for vitamin D is 200 lU (or cholecalciferol). Dietary sources Cood sourcesof vitamin D includefany fish, fish liver oils, egg yolk etc. Milk is not a good sourceof vitamin D. Vitamin D can be provided to the bodv in three ways '1. Exposureof skin to sunlightfor synthesis of vitamin D; 2. Consumptionof naturalfoods; 3. By irradiating foods (like yeast) that containprecursorsof vitamin D and fortification of foods (milk, butteretc.). Deficiency symptoms Vitamin D deficiency is relatively less common/sincethis vitamincan be synthesized in the body. However, insufficient exposure to sunlight and consumption of diet lacking vitamin D results in its deficiency. Vitamin D deficiency occurs in strict vegetar.ians,chronic alcoholics,individualswith Iiver and kidney diseasesor fat malabsorption syndromes.In somepeople,who coverthe entire body (purdah)for religiouscustoms,vitamin D deficiencyis alsoobserved,if the requirementis not met through diet. Deficiency of vitamin D causes rickets in children and osteomalacia in adults. derived from an old English *ord 'rvr-i.kk"n', meaning to twist. Osteomalacia is derived from Creek (osteon-bone; malakia-softness). Vitamin D is oftencalledasantirachiticvitamin. Ricketsin children is characterizedby bone deformitiesdue to incompletemineralization, resultingin soft and pliable bonesand delay in teeth formation.The weighrbearing bones are bent to lorm howJegs. In rickets, the plasma level of calcitriol is decreased and alkaline phosphatase activity is elevated. Alkaline phosphataseis concernedwith the processof bone formation.There is an overproductionof Plasmacalcium{ J India), 5mg t ,'i
  • 128 BIOCHEMISTRY alkalinephosphataserelatedto morecellular activitvof the bone. lt is believedto be due to a vain attemptto resultin boneformation. In case of osteomalacia(adult rickets) demineralizationof the bonesoccurs(bones becomesofter),increasingtheirsusceptibility to fractures. Rena! rickets {renal osteodystrophy) Fiq.7.10 : Structureof a-tocopherol(Note : The tocopherols differ in the substitution of methyl groups, represented in red). cH2-(cH2-cH2-cH-cH2)3-H cr,-Tocopherol(5,7,8-trimethyltocol) p-Tocopherol(5,8-dimethyltocol) y-Tocopherol(7,8-dimethyltocol) tocopherols (vitamin E vitamers) have been identified-a, p, T, 6 etc. Among these, u-tocopherol is the most active.The tocopherols are derivatives of 6-hydroxy chromane (tocol) ring with isoprenoid(3 units) side chain. The antioxidantpropertyisdue to the chromanering. Absorption, transport and storage Vitamin E is absorbedalong with fat in the small intestine.Bile saltsare necessaryfor the absorption.In the liver, it is incorporatedinto lipoproteins(VLDL and LDL) and transported. Vitamin E is stored in adiposetissue,liver and muscle.The normal plasmalevelof tocopherol is lessthan 1 mg/dl. Biochemical functions Most of the functionsof vitamin E are related to its anfioxidant property. lt preventsthe non- enzymaticoxidationsof variouscell components (e.g. unsaturated fatty acids) by molecular oxygen and free radicals such as superoxide (Otl and hydrogen peroxide (H2O2). Ttie element seleniumhelpsin thesefunctions. Vitamin E is lipophilic in characterand is found in association with lipoproteins, fat depositsand cellularmembranes.It protectsthe polyunsaturated fatty acids (PUFA) from peroxidation reactions. Vitamin E acts as a scavengerand gets itself oxidized (to quinone form) by free radicals(R) and sparesPUFA,as shown below Hsc. cHs CHn t- HO This is seenin patientswith chronicrenal failure.Renalricketsis mainly due to decreased synthesisof calcitriolin kidney.lt can be treated by administrationof calcitriol. Hypervitaminosis D Vitamin D is storedmostlyin liverand slowly metabolised.Among the vitamins,vitamin D is the mosf toxic in overdoses(10-100timesRDA). Toxic effects of hypervitaminosisD include demineralization of bone (resorption) and increasedcalcium absorptionfrom the intestine, Ieading to elevated calcium in plasma (hypercalcemia). Prolonged hypercalcemiais associatedwith depositionof calcium in many soft tissuessuch as kidney and arteries.Hyper- vitaminosisD may leadto formationof stonesin kidneys (renal calculi). High consumptionof vitamin D is associatedwith loss of appetite, nausea,increasedthirst, lossof weight etc. Vitamin E (tocopherol)is a naturallyoccurring antioxidant.lt isessentialfor normalreproduction in many animals,hence known as anti-sterility vitamin. Vitamin E is describedas a 'vitamin in searchof a disease.'Thisis due to the lackof any specificvitaminE deficiencydiseasein humans. Evansand his associates(1936) isolatedthe compounds of vitamin E activity and named them as tocopherols(Creek : tokos-childbirth; pheros-tobear;ol-alcohol). Clremistry Vitamin E is the name given to a group of tocopherols and tocotrienols. About eight
  • Chapter 7 : VITAMINS 129 The biochemical functions of vitamin E, related either directly or indirectly to its antioxidantproperty,are given hereunder 1. Vitamin E is essentialfor the membrane structureand integrityof the cell, hence it is regarded as a membrane antioxidant. 2, lt prevents the peroxidation of poly- unsaturatedfatty acids in various tissuesand membranes.lt protectsRBC from hemolysisby oxidizingagents(e.g.H2O2). 3. lt is closely associatedwith reproductive functions and prevents sterility. Vitamin E preservesand maintainsgerminalepitheliumof gonadsfor proper reproductivefunction. 4, lt increasesthe synthesisof heme by enhancing the activity of enzymes 6- aminolevulinicacid (ALA) synthaseand ALA dehydratase. 5. lt is required for cellular respiration- through electron transport chain (believed to stabilizecoenzymeQ). 6. Vitamin E prevents the oxidation of vitamin A and carotenes. 7. lt is requiredfor properstorageof creatine in skeletalmuscle. 8. Vitamin E is neededfor optimalabsorption of amino acids from the intestine. 9. lt is involvedin propersynthesisof nucleic acids. 10. Vitamin E protects liver from being damagedby toxic compoundssuch as carbon tetrachloride. 11, lt worksin associationwith vitaminsA, C and p-carotene,to delay the onset of cataract. 12. Vitamin E hasbeenrecommendedfor the preventionof chronic diseasessuch as cancer and heartdiseases.Clinical trials in this regard are rather disappointing,hence it is no more recommended. However, some clinicians continue to use it particularly in subjects susceptible to heart attacks. lt is believed that vitamin E preventsthe oxidation of LDL. (Note : The oxidized LDL have been implicated to promote heartdiseases.) Vitamin E and selenium Theelementseleniumisfoundin theenzyme glutathione peroxidase that destroys free radicals.Thus,Seisalsoinvolvedin antioxidant functionslike vitaminE, and bothof themact synergistically.To a certainextent,Secanspare the requirementvitaminE,andviceversa. Recommended dietary allowance (RDA) Intakeof vitaminE is directlyrelatedto the consumptionof polyunsaturatedfatty acids (PUFA)i.e.,requirementincreaseswith increased intakeof PUFA.A dailyconsumptionof about l0 mg(15lU)of c-tocopherolformanandI mg (12 lU)for womanis recommended.One mgof a-tocopherolis equalto 1.5 lU. VitaminE supplementeddiet is advisedfor pregnantand lactatingwomen. Dietary sources Many vegetableoils are rich sourcesof vitamin E. Wheat germ oil, cotton seedoil, peanutoil, cornoil and sunfloweroil arethe goodsourcesof thisvitamin.lt isalsopresentin meat,milk, butterandeggs. Deficiency symptoms The symptomsof vitaminE deficiencyvary from one animalspeciesto another.In many animals,the deficiency is associatedwith sterility, degenerativechanges in muscle, megaloblasticanaemiaand changesin central nervoussystem.Severesymptomsof vitaminE deficiencyare not seen in humansexcept increasedfragilityof erythrocytesand minor neurologicalsymptoms, Toxicity of vitamin E Amongthe fat solublevitamins(A, D, E, K), vitaminE is the leasttoxic. No toxic effecthas beenrepoftedevenafter ingestionof 300 mg/ dayfor 23 years. Vitamin K is the only fat solublevitaminwith a specificcoenzymefunction. lt is requiredfor
  • 130 BIOCHEMISTRY the production of blood clotting factors,essentialfor coagulation(in Cerman-Koagulation; hence the name K for this vitamin). Ghemistry Vitamin K exists in different forms (Fig.7.ll). Vitamin K1 (phylloquinone)is presentin plants. Vitamin K2 (menaquinone) is produced by the intestinalbacteria and alsofound in animals.Vitamin K3 (menadione)is a syntheticform. All the threevitamins(K1, K2,K3) are naphthoquinone derivatives. lsoprenoidside chain is presentin vitamins Kt and K2. The three vitamins are stable to heat. Their activity is, however, lost by oxidizing agents,irradiation,strong acidsand alkalies. Absorption, transport and storage CH2-CH:C-CH2- (CH2-CH2-CH-CH2)3-H VitamlnK1(phylloquinone) o CHs CH? t- VitaminK2(menaquinone) VitamlnK3(menadione) Fig.7.l1 : Structuresol vitaminK. ?r, Vitamin K is taken in the diet or svnthesized by the intestinalbacteria.Its absorptiontakes place along with fat (chylomicrons) and is dependenton bile salts.Vitamin K is transported alongwith LDLand is storedmainlyin liverand, to a lesserextent,in other tissues. Biochemical functions Thefunctionsof vitaminK areconcernedwith blood clotting process.lt bringsabout the post- translational (after protein biosynthesis in the cell) modification of certain blood clotting factors.The clotting factorsll (prothrombin),Vil, lX and X are synthesizedas inactiveprecursors (zymogens)in the liver. Vitamin K acts as a coenzyme for the carboxylation of glutamic acid residuespresentin the proteinsand this reaction is catalysedby a carboxylase(microsomal).lt involves the conversionof glutamate(Clu) to y-carboxyglutamate(Gla)and requiresvitamin K, 02 and COz Gig.7.l. The formation of y-carboxyglutamate is inhibited by dicumarol, an anticoagulantfound in spoilt sweet clover. Wartarinis a syntheticanaloguethat can inhibit vitamin K action (Fig.7.13). Vitamin K is also required for the carboxylation of glutamic acid residues of osteocalcin,a calcium binding protein present in the bone. The mechanismof carboxylationis not fully understood. lt is known that a 2,3-epoxide derivative of vitamin K is formed as. an intermediateduring the courseof the reaction. Dicumarol inhibitsthe enzyme (reductase)that convertsepoxideto active vitamin K. Role of Gla in clotting : The lcarboxy- glutamic acid (Cla) residuesof clotting factors are negatively charged (COO-) and they combine with positivelychargedcalcium ions (Ca2+)to form a complex. The mechanismof action has been studied for prothrombin.The prothrombin -Ca complex binds to the phospholipidson the membranesurfaceof the platelets(Fig.7,14. This leadsto the increased conversionof prothrombinto thrombin. Recommended dietary allowance (RDA) Strictlyspeaking,there is no RDA for vitamin K, since it can be adequatelysynthesizedin the
  • Ghaprer 7 : VITAMINS 131 H I Protein,^.,,',2,,rN-CH- C/^,/"v',, lll cH2o Glu--Jl cHz I cooH Precursorsof clotting lactors(ll,Vll,lX,X) H I PrOtein,^:,,T,2:,,rNl- CH- C,^,4/',, lll cH2o I CH{-Gla -<4]---cooH cooH Clotting factors (ll, Vll, lX, X) VitaminK coz T ?Dicumarol, warfarin Fig. 7.12 : Vitamin K dependent carboxylation of the precursors of clotting factors. gut. lt is however,recommendedthat half of the body requirementis provided in the diet, while the otherhalf is metfrom the bacterialsynthesis. Accordingly,the suggestedRDA for an adult is 7o-140 ttg/day. Dietary sources Cabbage, cauliflower, tomatoes, alfa alfa, spinach and other green vegetablesare good sources.lt is alsopresentin eggyolk, meat,liver, cheeseand dairy products. Deflciency symptoms The deficiency of vitamin K is uncommon, sinceit is presentin the diet in sufficientquantity and/oris adequatelysynthesizedby the intestinal bacteria. However, vitamin K deficiency may occur due to its faulty absorption(lack of bile salts), loss of vitamin into feces (diarrheal diseases)andadministrationof antibiotics(killinS of intestinalflora). Deficiencyof vitamin K leadsto the lack of activeprothrombinin the circulation.The result is that blood coagulationis adverselyaffected. The individualbleedsprofuselyeven for minor injuries. The blood clotting time is increased. Hypervitaminosis K Administrationof large doses of vitamin K produces hemolytic anaemia and jaundice, particularlyin infants.The toxic effectis due to increasedbreakdownof RBC. Antagonists of vitamin K Thecompounds-namelyheparin,bishydroxy- coumarin-act as anticoagulants and are antagoniststo vitamin K. The salicylatesand dicumarol are also antagoniststo vitamin K. (Protein)-Glu (Protein)-Gla H -N-CH-C- lrl QHzo I -ctt.tt O=C C=O tltt ll -o o- "".Cd2* -r=r'#-x YYYYYYYY AAAAAAAA Prothrombin VitaminK (hydroquinoneform) Redilcta$e _ 2,3-Epoxideform I/ neductase ,/ Quinone marol, torm r{arin y-Carboxyglutamate complexedwithcalcium Plateletmembrane (withphospholipids) Fiq.7.13 : Summaryof vitaminK cycle in carboxylation reactian. Fiq.7"14: MechanismofactionofTcarboxyglutamate tE
  • 132 BIOCHEMISTRY O=Q J. + H2O Iv- O=C I =?-l H-?6 H-? |_Cr I HO-C-H I tl cH2oH cH2oH D'ahydroL-ascorbic DlketoL€ulonlc acld(oxidizedtorm) acid dehydroascorbic acid are biologically active. However, D-ascorbicacid is inactive.The plasma and tissues pre- dominantly contain ascorbic acid in the reducedform. The ratio of ascorbic acid to dehydroascorbicacid in many tissuesis 15 : 1. On hydration, dehydroascorbic acid is irreversiblyconverted to 2,3- diketogulonic acid which is inactive. Hvdration reaction is O=C-OH O=C I O:C I H-C-OH I HO-C-H cooH cooH cH2oH L-Ascorblcacid (reducedform) Oxalic acld Fiq.7.15: Sttucturesof vitaminC (ascorbicacid) anditsrelatedcompounds. Dicumarolis structurallyrelatedto vitaminK and almost spontaneous,in alkaline or neutral actsas a competitiveinhibitorin the synthesisof solution. lt is for this reasonthat oxidation of activeprothrombin. vitamin C is regardedas biological inactivation (formationof diketogulonicacid). Oxidation of ascorbicacid is rapid in the presenceof copper, Hence vitamin C becomesinactiveif the foods are preparedin copper vessels. Biosynthesis and metabolism Many animals can synthesize ascorbic acid from glucose via uronic acid pathway (Chapter 13). Howevet, mant other primates, guineapigsand batscannotsynthesizeascorbic acid due to the deficiency of a single enzyme namely L-gulonolactoneoxidase. Vitamin C is rapidly absorbed from the intestine, lt is not stored in the bodv to a significantextent. Ascorbic acid is excretedin urine as such, or as its metabolites- diketogulonicacid and oxalic acid (Fig,7,1fl. Biochemical functions Most of the functionsof vitamin C are related to its propertyto undergoreversibleoxidation- reduction i.e., interconversionof ascorbicacid and dehydroascorbicacid. 1, Collagenformation : Vitamin C playsthe role of a coenzymein hydroxylationof proline and lysine while protocollagenis convertedto collagen (i.e. post-translational modification). The hydroxylationreactionis catalysedby lysyl hydroxylase (for lysine) and prolyl hydroxylase (for proline). This reaction is dependent on vitaminC, molecularoxygenand a-ketoglutarate (Fig.7.t6). VitaminC is a watersolubleversatilevitamin. It playsan importantrole in human healthand disease. Vitamin C has become the most controversialvitamin in recent years.This is becauseof the claimsand counter-claimson the useof vitaminC in megadosesto cureeverything from common cold to cancer. Scurvyhasbeen known to man for centuries. It was the first diseasefound to be associated with diet. In the sixteenthcenturyabout 10,000 marinersdied of a miraculousdisease(scurvy) due to lack of fresh vegetablesin their diet. JamesLind, a surgeonof the EnglishNavy, in 1753 published'Treatiseon Scurvy'.Basedon Lind'sobservations,the RoyalNavy since1795 used to supply lime or lemon juice to all the crews.The EnglishNavy usedto carry cratesof lemons, hence they were popularly known as Limeys. Ghemistry Ascorbic acid is a hexose (6 carbon) derivative and closelv resemblesmono- saccharidesin structure(Fig.7.15).The acidic propertyof vitamin C is due to the enolic hydroxyl groups. lt is a strong reducing agent. L-Ascorbicacid undergoesoxidation to form dehydroascorbicacid and this reactionis reversible.Both ascorbicacid and
  • Chapter 7 : VITAMINS 133 HO ,,'.",,.."z".,,"'.il-cH-8,,"'.,,"..,,".",". I IIHeC.- .CHzl- Proline c- I H2 I a-Ketoglutara," l r,-O, V Prolylhydroxylase Ascorbicacid /| ,/l Succinate+CO2aJt"rg HO ,..,..,,-N-oH-f* HeC- ,/ +-Hydroxyproline- n/' Hr''" Fig. 7.16 : Ascorbicacid dependent hydroxylation of proline of protocollagen. Hydroxyproline and hydroxylysine are essentialfor the collagencross-linkingand the strengthof the fiber. In this way, vitamin C is necessaryfor maintenanceof normalconnective tissueand wound healingprocess. 2. Bone formation : Bone tissuespossessan organic matrix, collagen and the inorganic calcium, phosphateetc. Vitamin C is required for bone formation. 3. lron and hemoglobin metabolism: Ascorbic acid enhances iron absorption by keepingit in the ferrousform. This is due to the reducingpropertyof vitamin C. lt helps in the formation of ferritin (storageform of iron) and mobilizationof iron from ferritin. Vitamin C is useful in the reconversionof methemoglobinto hemoglobin.The degradation of hemoglobinto bile pigmentsrequiresascorbic acid. 4. Tryptophan metabolism: Vitamin C is essentialfor the hydroxylation of tryptophan (enzyme-hydroxylase)to hydroxytryptophanin the svnthesisof serotonin. 5. Tyrosine metabolism: Ascorbic acid is required for the oxidation of p-hydroxy phenylpyruvate(enzymehydroxylase)to homo- gentisicacid in tyrosinemetabolism. 6. Folic acid metabolism : The active form of the vitamin folic acid is tetrahydrofolate(FHr). Vitamin C is neededfor the formation of FHa (enzyme-folic acid reductase). Further, in associationwith FHz, ascorbicacid is involved in the maturation of erythrocytes. 7. Peptidehormone synthesis: Many peptide hormones contain carboxyl terminal amide which is derived from terminal glycine. Hydroxylation of glycine is carried out by peptidylglycine hydroxylase which requires vitamin C. 8. Synthesis of corticosteroid hormones : Adrenal gland possesseshigh levelsof ascorbic acid, particularly in periods of stress. lt is believed that vitamin C is necessaryfor the hydroxylation reactions in the synthesis of corticosteroidhormones. 9. Sparing action of other vitamins : Ascorbic acid is a strongantioxidant.lt spares vitamin A, vitamin E, and some B-complex vitaminsfrom oxidation. 10. lmmunological function : Vitamin C enhances the synthesisof immunoglobulins (antibodies)and increasesthe phagocyticaction of leucocytes. 11. Preventiveaction on cataract: Vitamin C reducesthe risk of cataractformation. 12. Preventiveaction on chronic diseases: As an antioxidant,vitamin C reducesthe risk of cancer, cataract, and coronary heart diseases. Recommended dietary allowance (RDA) About 60-70 mg vitamin C intake per day will meet the adult requirement.Additional intakes Q0-4O%increase)are recommendedfor women during pregnancyand lactation.
  • 134 BIOCHEMISTPY Slietary sourees Citrusfruits,gooseberry(amla),guava/green vegetables (cabbage, spinach), tomatoes, potatoes(particularlyskin) are rich in ascorbic acid. High content of vitamin C is found in adrenalglandand gonads.Milk is a poor source of ascorbicacid. tsefiaiem*y symptoms The deficiencyof ascorbic acid resultsin scurvy. This diseaseis characterizedby spongy and sore gums, Ioose teeth, anemia, swollen joints, fragile blood vessels, decreased immunocompetence,delayed wound healing, sluggishhormonalfunctionof adrenalcortexand gonads,haemorrhage,osteoporosisetc. Most of thesesymptomsare relatedto impairmentin the synthesisof collagen and/or the antioxidant propertyof vitamin C. Megadoses of nitannEm C afid it$ r}Gntrobrersy Linus Pauling (1970) first advocated the consumptionof megadosesof ascorbic acid (even up to 18 g/day, 300 times the dailv requirement!) to prevent and cure common cold. He is remembered as a scientist who suggested'keepvitaminC in gunnybagsand eat in grams.'This generateda lot of controversy' worldover. lt is now clear that megadoseof vitamin C does not oreventcommon cold. But the duration of cold and the severity or symptoms are reduced. lt is believed that ascorbicacid promotesleukocytefunction. Megadoses(-a ildaV) of vitamin C are still continued in common cold, wound healing, trauma etc. As an antioxidant,ascorbicacid certainlyprovidessomehealthbenefits. Ascorbicacid,assuch,hasnot beenfoundto be toxic. But, dehydroascorbicacid (oxidized form of ascorbicacid)istoxic.Further,oxalateis a major metaboliteof vitamin C. Oxalate has been implicated in the formation of kidner stones.However,there are controversialreports on the megadosesof vitaminC leadingto urinarl stones. BIOMEDIGALI CLINICALCONGEPTS It is belieuedthat during the courseof euolution,the ability to synthesizeuitaminsuos lost by the higher orgonisms,hence they should be supplied through the diet. For humans,the normal intestinalbacterialsgnfhesisoJ uitamin K and biotin is olmost sufficientto meet the bodg requirements. Administrationot' antibioticsolten destroysthe uitamin synthesizingbacteriain the gut, henceadditionalsupplementationot' uitaminsis recommendedduring antibiotictherapy. Vitamin A det'iciencqcousesnight blindness;uifomin D deficiency rickets (in children) or osteomalacia(in adults); uitamin E deficiencyminor neurologicalsymptoms;uitamin K deficiencybleeding. Fat soluble uifomins are not readily excreted in urine, hence excesscohsumption leads to their accumulation qnd toxic effects. Vitomin C deliciency cdusesscuruy. The monifestotionsoJ scuruyore related to the impairment in the synthesisoJ collagenand/or the antioxidant property of uitomin C. ' Megadosesof uitomin C are used in common cold, wound healing,trauma etc. ftCarotene, uitamin E and ascorbicacid serueos entioxidantsand reduce the risk oJ heart sttacks and cancers,
  • Ghapter 7 : VITAMINS 135 Reactive 2. cr-Ketoglutaratedehydrogenase carbon NHz J _ ls an enzymeof the citric acid cycle. This enzvme is comparable withI nts enzyme ts comparable with I pyruvatedehydrogenaseand requires )-p O- TPP. O , 3. Transketolaseis dependenton +l# rrvv'v"v ------f----- | --T ---l-------- TPP. This is an enzyme of the hexose PyrimidineMethylene Thiazole Pyrophosphate monophosphateshunt (HMp shunt). bridge 4. The branched chain a-ketoThiamine acid dehydrogenase (decarboxylase) Flg.7.17: Structuresofthiamineandthiamine catalysesthe oxidativedecarboxylation pyrophosphate(TPP)' of branchedchain amino acids(valine, leucine and isoleucine) to the respectiveketo acids.This enzymealso requires TPP. 5. TPP plays an important role in the transmissionof nerve impulse.lt is believedthat TPP is requiredfor acetylcholinesynthesisand the ion translocationof neuraltissue. Flecornnrended dietary a!iswanee {RDA} The daily requirementof thiaminedepends on the intakeof carbohydrate.A dietarysupply ol 1-1.5 mdday is recommendedfor adults (about0.5 hrg/1,000Calsof energy).Forchildren RDA is 0.7-1.2 m{day. The requirement marginallyincreasesin pregnancyand lactation (2 mglday),old age and alcoholism. Dietary souree$ Cereals,pulses,oil seeds,nuts and yeastare good sources.Thiamine is mostly concentrated in the outer layer(bran)of cereals.Polishingof rice removesabout80% of thiamine.Vitamin B, is also presentin animal foods like pork, liver, heart,kidney,milk etc. ln the parboiled(boiling of paddywith husk)and milled rice,thiamineis not lost in polishing.Sincethiamineis a water soluble vitamin, it is extractedinto the water during cookingprocess.Suchwater should not be discarded. Deliciency $ymptonns The deficiency of vitamin B1 results in a condition called beri-beri lsinhalese : I cannot Thiamine (anti-beri-beri or antineuritic vitamin) is water soluble. lt has a specific coenzyme/ thiamine pyrophosphate (TPP)which is mostly associated with carbohydrate metabolism. Chenristry Thiaminecontainsa pyrimidinering and a thiazole ring held by a methylene bridge (Fig.7.1V. Thiamine is the only natural compoundwith thiazolering. The alcohol (OH) group of thiamine is esterfiedwith phosphate(2 moles)to form the coenzyme, thiamine pyrophosphate (TPP or cocarboxylase). The pyrophosphate moiety is donated by ATP and the reaction is catalysed by the enzyme thiamine pyrophosphate transferase. 9it:chemical funetiens The coenzyme, thiamine pyrophosphateor cocarboxylaseis intimatelyconnectedwith the energy releasingreactionsin the carbohydrate metabolism(FiS.7.lA. 1. The enzyme pyruvate dehydrogenase catalyses (oxidative decarboxylation) the irreversibleconversion of pyruvate to acetyl CoA. This reactionis dependenton TPP,besides the other coenzymes (details given in carbohydratemetabolism,Chapter 13). *h
  • 136 BIOCHEMISTF|Y Glucose ------+ Glucose6-phosphate Pyruvate Ribose5-phosphate Oxaloacetate Citrate Fi4.7.18: Summaryofthereactionsdependentonthiaminepyrophosphate(TPP). (said twice)1. Beri-beri is mostly seen in populationsconsumingexclusivelypolishedrice as staplefood. The early symptomsof thiamine deficiency are loss of appetite (anorexia), weakness, constipation, naLrsea, mental depression, peripheral neuropathy, irritability etc. Numbnessin the legs complaintsof 'pins and needlessensations'are reported. Biochemical changes in B'' deficiency 1. Carbohydratemetabolism is impaired. AccumulationoI pyruvate occurs in the tissues which is harmful. Pyruvate concentration in plasmaiselevatedand it is alsoexcretedin urine. 2. Normally, pyruvate does not cross the blood-brain barrier and enter the brain. However, in thiamine deficiency,an alteration occurs in the blood-brainbarrierpermittingthe pyruvateto enterthe braindirectly.lt is believed that pyruvateaccumulation in brain resultsin disturbed metabolismthat may be responsible for polyneuritis. 3. Thiaminedeficiencyleadsto impairment in nerveimpulsetransmissiondue to lackof TPP. 4. Thetransketolaseactivityin erythrocytesis decreased.MeasuremenLof RBC transketolase activity is a reliable diagnostic test to assess thiaminedeficiencv. In adults,two typesof beri-beri,namelywet beri-beriand dry beri-berioccur. lnfantileberi- beri that differsfrom adult beri-beriis alsoseen. Wet beri-beri: This is characterized by edema of legs, face, trunk and serouscavities. Breathlessnessand palpitationare present.The calf musclesare slightlyswollen.The systolic blood pressureis elevated while diastolic is decreased.Fastand bouncingpulseis observed. The heart becomesweak and death may occur due to heartfailure. Dry beri-beri : This is associatedwith neuro- Iogical manifestations resulting in peripheral neuritis. Edema is not commonly seen. The musclesbecomeprogressivelyweak and walking
  • Chapter 7 : VITAMINS 137 becomes difficult. The affected individuals depend on support to walk and become bedridden,and may even die if not treated. The symptomsof beri-beriare often mixed in which case it is referredro as mixed beri-beri. Infantile beri-beri: This is seen in infants born to mothers suffering from thiamine deficiency. The breast milk of these mothers containslow thiaminecontent.Infantileberi-beri is characterizedby sleeplessness,restlessness, vomiting,convulsionsand bouts of screaming that resembleabdominal colic. Most of these symptomsare due to cardiac dilatation.Death may occur suddenlydue to cardiacfailure. Wernicke-Korsakoff syndrome This is a disorder mostlv seen in chronic alcoholics. The body demands of thiamine increasein alcoholism. lnsufficientintake or impairedintestinalabsorptionof thiaminewill leadto this syndrome.lt is characterizedby loss of memory,apathyand a rhythmicalto and fro motion of the eye balls. Thiamine deficiency due to thiaminase and pyrithiamine The enzyme thiaminaseis presentin certain seafoods.Their inclusionin the diet will destrov thiamineby a cleavageaction (pyrimidineand thiazole rings are separated) and lead to deficiency.Pyrithiamine,a structuralanalogue and an antimetaboliteof thiamine;is found in certainplantslike ferns.Horsesand cattleoften develop thiamine deficiency(fern poisoning) due to the overconsumptionof the plant fern. Thiamine antagonists Pyrithiamine and oxythiamine are the two importantantimetabolitesof thiamine. Riboflavinthroughits coenzymestakespart in a varietyof cellularoxidation-reduction reactions. Chemistry Riboflavin contains 5,7-dimethyl isoalloxazine (a heterocyclic 3 ring structure)attached to D-ribitol by a nitrogenatom. Ribitol is an open chain form of sugar ribose with the aldehyde group (CHO) reducedto alcohol (CH2OH). Riboflavin is stable to heat but sensitiveto light. When exposed to ultra-violet rays of sunlight, it is converted to lumiflavin which exhibits vellow fluorescence.The substances namely lactoflavin (from milk), hepatoflavin (from liver) and ovoflavin (from eggs) which were originally thought to be different are structurallyidenticalto riboflavin. Coenzymes of riboflavin Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the two coenzyme forms of riboflavin. The ribitol (5 carbon)is linkedto a phosphatein FMN. FAD is formed from FMN by the transferof an AMP moiety from ATP (Fig,7.19/'. Biochemical functions The flavin coenzymes(mostlyFAD and to a lesserextent FMN) participatein many redox reactionsresponsiblefor energyproduction.The functional unit of both the co€rZln,rs is isoalloxazineringwhich servesasan acceptorof two hydrogen atoms (with electrons).FMN or FAD undergo identical reversible reactions acceptingtwo hydrogenatomsforming FMNH2 or FADH2 ffi9.7.20). Enzymesthat useflavin coenzymes(FMN or FAD) are called flavoproteins. The coenzymes (prostheticgroups)often bind rather tightly, to the protein(apoenzyme)either by non-covalent bonds (mostly) or covalent bonds in the holoenzyme.Many flavoproteinscontain metal atoms(iron,molybdenumetc.)which areknown as metallofl avoproteins. The coenzymes,FAD and FMN areassociated with certainenzymesinvolved in carbohydrate, lipid, protein and purine metabolisms,besides the electrontransportchain.A few examplesare listed in Table7,2. Furtherdetailsare given in the respectivechapters,
  • 135 BIOCHEMISTF etmfy quirement of tlt is 1.2-1,7mg. 0.2-0.5 mdday) )r pregnant and ,'ees Milk and milk products,meat, eggs,liver, kidney are rich sources. Cereals,fruits, vegetablesand fish are moderate sources. Defieienclr symptonrs Riboflavin deficiency symptoms include cheilosis (fissuresat the corners of the mouth), glossitis (tongue smooth and purplish) and dermatitis. Riboflavin deficiency as suchis uncommon.lt is mostlyseen along with other vitamin defi- ciencies. Chronic alcoholics are susceptibleto 82 deficiency.Assay of the enzyme glutathione reductase in erythrocyteswill be useful in assessingriboflavindeficiency. Antimetabolite: Calactoflavinis an antimetaboliteof riboflavin. Niacin or nicotinic acid is also known as pellagra preventive (P.P.) factor oI Coldberg.The coenzymes of niacin (NAD+ and NADP+)can be synthesized by the essential amino acid, tryptophan. The disease pellagra (ltalian : rough skin) has been known for centuries.However, its relation to the deficiencyof a dietaryfactorwas first identified by Coldberger. Coldberger and his associates conductedan interestingexperiment o ll H_C_OH T lo cH2oH L Riboflavin Flavoklnasei o CH, I- H-C-OH I H-C-OH I H-C-OH O lll cH2o-P-o--6 Flavinmononucleotide(FMN) Dsvn_thgp.e, o 9HzI H-C-OH I H-C-OH I H-C-OHO O o- o- Flavin adenlne dlnucleotlde (FAD) Fig. 7,19: Structuresand biosynthesisof flavinmononucleotide (FMN)and flavinadeninedinucleotide(FADL
  • Ghapter 7 : VITAMINS 139 I @ Oxidizedflavin (FMNor FAD) @ Reduced flavin (FMNH2or FADH2) I H Fig, 7,20 : Pariicipation of FMN or FAD in oxidation-reduction reactions (R-represents the rest of the structurcof FMN or FADas depictedin Fiq.7.19). for this purpose.Twelveconvictswere promised pardon if they consumed diet of pellagrous familiesfor one year.The diet consistedof corn meal/ corn starch, rice, sweet potato and pork fat. More than half of the subjects showed symptomsof pellagrain lessthan an year,while no such symptoms were observed in other prisonerson a regulardiet. Administrationof dried meator liverto the patientscured pellagra (Coldberger, 1928). Much before it was recognizedas a vitamin, nicotinicacid was well known as a chemical compound, produced by the oxidation of nicotine (presentin tobacco leaves).The term 'niacin' was coined and more commonlv used for nicotinicacid. This was done to emohasize the role of niacin as a vitamin and avoid the impressionthat nicotinic acid is an oxidized form of nicotine.However, most of the authors use niacin and nicotinicacid synonymously. GhemistrV amd snrmthesis of coenzymes Niacin is a pyridine derivative. Structurally,it is pyridine3-carboxylicacid.Theamideform of niacinis knownas niacinamideor nicotinamide. Enzyme Reaction FADdependent l" Carbohydratemetabolism (a)Pyruvatedehydrogenasecomplex* (b)cr-Ketoglutaratedehydrogenasecomplexx (c)Succinatedehydrogenase ll. Lipidmetabollsm (d)AcylCoAdehydrogenase lll. Proteinmetabolisn (e)Glycineoxidase (f)D-Aminoacidoxidase lY. Purinemetabolism (g)Xanthineoxidase Pyruvate------+AcetylCoA a-Ketoglutarate------+SuccinylCoA Succinate-----+Fumarate AcylCoA---+ o, B-UnsaturatedacylCoA Glycine-+ Glyorylate+ NH3 D-Aminoacid-> cl-Ketoacid+ NH3 Xanthine------+Uricacid FMN dependent L-Aminoacidoxidase * DihydrolipoyldehydrogenasecomponentrequiresFAD L-Aminoacid---------+a-Ketoacid+ NHo .
  • 140 BIOCHEMISTRY Dietary nicotinamide,niacin and tryptophan (an essentialamino acid) contribute to the synthesis of the coenzymes-nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+)" The pathway for the biosynthesisof NAD+ and NADP+ is depictedin Fi9.7.21.Nicotinamideis deaminatedin the bodv to niacin. Niacin then undergoesa seriesof reactionsto produceNAD+ and NADP+. Tryptophan producesquinolinate which then forms nicotinate mononucleotide and, ultimately, NAD+ and NADP+. Sixty milligrams of tryptophan is equivalent to I mg of niacin for the synthesis of niacin coenzymes. Phosphoribosylpyrophosphateand ATP, respectively,provideribosephosphateand AMP moieties for the synthesis of NAD+. Clutaminedonatesamidegroup.ln the structure of the coenzymes,nitrogenatom of nicotinamide carriesa positivecharge(dueto the formationof an extra bond, N is in quaternarystate),hence the coenzymes are NAD+ and NADP+. Nicotinamide, liberatedon the degradationof NAD+ and NADP+ is mostlv excretedin urine as N-methyInicoti namide. (Note : Some authors prefer to use NAD/ NADP without a positive charge to represent generalor oxidized form of niacin coenzymes.) Biochemical functions The coenzvmes NAD+ and NADP+ are involved in a variety of oxidation-reduction reactions.They accept hydride ion (hydrogen atom and one electron :H-) and undergo reductionin the pyridinering.Thisresultsin the neutralizationof positivecharges.The nitrogen atom and the'fourth carbon atom of nicotinamidering participatein the reaction. While one atom of hydrogen(as hydride ion) from the substrate(AH2) is accepted by the coenzyme, the other hydrogen ion (H+) is releasedinto the surroundingmedium. This reaction is reversedwhen NADH is oxidized to NAD+. NADP+ also functionslike NAD+ in the oxidation-reductionreactions. A large number of enzymes (about 40) belonging to the class oxidoreductases are loH isphosphorylated(-O-PO3-)l Fiq.7.21 : Outlineof the biosynthesisof nicotinamide nucleotides,NAD"and NADP (NPRT-Nicotinate Nicotinamide -l ll ------+rryprophan s2 -N- Nlacin i IN-Methyl- nicotinamide (excreted in urine) Nicotinate mononucleotide adenyltransferase Desamido-NAD* NADP+ Nicotinamide
  • chapter 7 : VITAMINS 141 H .Z>coNH2 il -'--i2 Ribose Adenine I @-@-Ribose NAD'(oxidized) Overallreaction amino acid tryptophancan serveas a precursor for the synthesisof nicotinamidecoenzymes.On an average, i g of a good quality protein containing about 60 mg of tryptophan is equivalent to 1 mg of niacin (conversionratio 60: 1) for the synthesis of nicotinamide coenzymes.Tryptophanhasmanyotheressential and important functions in the body, hence dietary tryptophan cannot totally replaceniacin. Increasedconversionof tryptophanto niacin has beenreportedin low proteindiet and starvation. Tryptophancan replace niacin to an extent of 10o/ofor the synthesisof coenzymes.Therefore, both niacinand tryptophanhaveto be invariably provided in the diet. Deficiency symptonns Niacindeficiencyresultsin a conditioncalled pellagra (ltalian: rough skin). This disease involvesskin, gastrointestinaltract and central nervoussystem.The symptomsof pellagraare commonly referredto as fhree Ds. The disease also progressesin that order dermatitis,diarrhea, dementia,and if not treated may rarely lead to death (4th D). Dermatitis(inflammationof skin) is usually found in the areasof the skinexposedto sunlight (neck,dorsalpartof feet,ankleand partsof face). Diarrheamay be in the form of loosestools,often with blood and mucus.Prolongeddiarrhealeads to weight loss. Dementia is associatedwith degenerationof nervoustissue.The symptomsof dementia include anxiety, irritability, poor memory,insomnia(sleeplessness)etc. Pellagrais mostlyseenamong peoplewhose staplediet is corn or maize. Niacin presentin maizeis unavailableto the bodvas it is in bound form. Further, tryptophan content is low in maize. Therapeutic uses of niaein Administrationof niacin in pharmacological doses(2-4 g/day,2OOtimes the RDA) resultsin a numberof biochemicaleffectsin the body, not relatedto its function as a vitamin. Most of the effectsare believedto be due to the influenceof niacinon cyclic AMP levels. coNH2-+ H+ I Ribose Adenine @-@-nibose NADH+ H+(reduced) AH2+NAD+HA+NADH+H+ Fiq.7.22 : Mechanismol oxidationand reductionof nicotinamide coenzyme-N AU (Note : Similar mechanism operates for NADP also). dependenton NAD+ or NADP+.The coenzymes are looselybound to the enzymesand can be separatedeasilyby dialysis.NAD+ and NADP+ participate in almost all the metabolisms tcarbohydrate,lipid, proteinetc.).Someenzymes are exclusively dependent on NAD+ whereas some requireonly NADP+.A few enzymescan useeither NAD+ or NADP+.Selectedexamoles of enzymesand the reactionsthey catalyseare given in Table7.3. NADH produced is oxidized in the electron transportchain fo generateATP. NADPH is also important for many biosynthetic reactionsas it donatesreducingequivalents. Recommended dietary allowance {BDAI Thedaily requirementof niacinfor an adultis 15-20mg and for children,around 10-15 mg. Very often, the term niacin equivalents(NE) is usedwhile expressingits RDA. One NE- 1 mg niacin or 60 mg of tryptophan.Insteadof mg, the daily requirementsare known as niacin equivalents.Pregnancyand lactationin women impose an additional metabolic burden and increasethe niacin requirement. Dietary source$ The rich natural sourcesof niacin include liver, yeast,whole grains,cereals,pulseslike beans and peanuts. Milk, fish, eggs and veeetablesare moderatesources.The essential
  • 142 BIOCHEMISTFIY Enzyme Reaction I NAD+ dependent L Carbohydratemetabolism (a) Glyceraldehyde3-phosphatedehydrogenase (b) Lactatedehydrogenase (c) Pyruvatedehydrogenasecomplex (d) a-Ketoglutaratedehydrogenasecomplex ll. Lipid metabolism (e) B-HydroxyacylCoAdehydrogenase (f) p-Hydroxybutyratedehydrogenase (g) Alcoholdehydrogenase lll. Proteinmetabolism (h) Branchedchainc-ketoaciddehydrogenase (i) Tyraminedehydrogenase NAD+ or NADP+dependent (a) Glutamatedehydrogenase (b) lsocitratedehydrogenase NADP+dependent (a) Glucose6-phosphatedehydrogenase Glyceraldehyde3-phosphate-----+1,3-Bisphosphoglycerate Pyruvate-----+Lactate Pyruvate------+AcetylCoA cr-Ketoglutarate----+ SuccinylCoA B-HydroxyacylCoA------+B-KetoacylCoA p-Hydroxybutyrate----+ Acetoacetate Alcohol--+ Acetaldehyde cr-Ketoacidsofbranchedchainaminoacids (Leu,lle,Val)-----+ConespondingacylCoAthioesters Tyramine----.>p-Hydroxyphenylacetate Glutamate------+a-Ketoglutarate+ NH, lsocitrate-----+Oxalosuccinate GlucoseG-phosphate-----+6-Phosphogluconolactone Malate---+ Pyruvate(b) Malicenzyme NADPHdependent (a) 3-Ketoacylreductase (b) HMGCoAreductase (c) Squaleneepoxidase (d) Cholesterol7a-hydrorylase (e) Phenylalaninehydroxylase (f) Dihydrofolatereductase 3-Ketoacylenzyme--> 3-Hydroxyacylenzyme HMGCoA--+ Mevalonale Squalene----+Squaleneoxide Cholesterol---+ 7o-Hydroxycholesterol Phenylalanine-----+Tyrosine Folicacid---+ Tetrahvdrofolicacid. 1. Niacin inhibitslipolysisin the adipose tissueand decreasesthe circulatoryfree fatty acids. 2. Triacylglycerolsynthesisin the decreased. 3. The serum levels of low density lipoproteins(LDL),very low densitylipoproteins (VLDL), triacylglycerol and cholesterol are lowered.Hence niacin is used in the treatment of hyperlipoproteinemia type II b (elevation of LDL and VLDL). Although megadosesof niacin are usefulfor the treatmentof hyperlipidemia,therearecertain harmful side effectsalso. 'l. Clycogen and fat reservesof skeletaland cardiacmuscleare depleted. 2. Thereis a tendencyfor the increasedlevels of glucoseand uric acid in the circulation.
  • 143 Chapter 7 : VITAMINS 3. Prolonged use of elevated serum levels of suggestingliver damage. niacin results in certain enzymes/ Vitamin 86 is used to collectivelY represent the three compounds namely pyridoxine, pyridoxal and pyridoxamine (the vitamers of 85,). Chemistry Vitamin 86 comPounds are PYridine derivatives.They differ from each other in the structureof a functionalgroup attached to 4th carbonin the pyridinering.Pyridoxine isa primaryalcohol,pyridoxalis an aldehyde form while pyridoxamine is an amine (Fig.7.23).Pyridoxamineis mostlypresentin plantswhile pyridoxaland pyridoxamineare found in animal foods. Pyridoxinecan be converted to pyridoxal and pyridoxamine, but the lattertwo cannotform pyridoxine' Symtlresis o{' coenzYme The active form of vitamin 86 is the coenzyme pyridoxal phosphate (PLP). PLP can be synthesizedfrom the threecompounds pyridoxine,pyridoxaland pyridoxamine'86 is excretedin urine as 4-pyridoxicacid. The different forms of 86 and their inter- relationshipare depictedin Fig.7.23. #icchemical {unetEons Pyridoxalphosphate(PLP),the coenzymeof vitamin 86 is found attached to the e-amino group of lysine in the enzyme' PLP is closely associated with the metabolism of amino acids. The synthesis of certain specialized products such as serotonin, histamine, niacin coenzymesfrom the amino acids is dependenton pyridoxine' Pyridoxalphosphate participates in reactions like transamination, decarboxyl ation, deamination, transsulfuration, condensation eIc. HO HsC cH2oH Pyridoxal ptto6ph# Pyridoxine tl'PYridoricacid Fiq.7.23 : Pyridoxine, its derivatives and coenzyme. 1. Transamination: Pyridoxal phosphateis involved in the transamination reaction (by transaminase)converteingamino acids to keto acids.The keto acidsenterthe citric acid cycle and get oxidized to generateenergy.Thus 86 is an energy releasing vitamin. lt integrates carbohydrate and amino acid metabolisms (Fig.7.24). During the course of transamination,PLP interactswith amino acid to form a Schiff base (Fig.7.25).The amino group is handed over to PLP to form pyridoxaminephosphateand the keto acid is liberated.
  • 144 BIOCHEMISTFIY 2. Decarboxylation: Some of the cr-amino acids undergo decarboxylation to form the respectiveamines.This is carriedout by a group of enzymes called decarboxylaseswhich are dependenton PLP.Many biogenicamineswith important functions are synthesized by PLP decarboxylation. (a) Serotonin (S-hydroxytryptamine,5 HT), producedfrom tryptophanis importantin nerve impulse transmission(neurotrans- mitter). lt regulates sleep, behaviour, blood pressureetc. Tryptophan-----+ 5-Hydroxytryptophan Coz 5-Hydroxytryptamine (b) Histamine is a vasodilator and lowers blood pressure.lt stimulatesgastricHCI secretionand is involvedin inflammation and allergicreactions. Histidine Histamine coz (c) Glutamate on decarboxylation gives yamino butyric acid (CABA). CABA inhibits the transmission of nerve impulses,hence it is an inhibitoryneuro- transmitter. Glutamate GABA coz (d) The synthesisof catecholamines(dopamine, norepinephrine and epinephrine) from tyrosine require PLP. Catecholamines are involved in metabolic and neryous regulation. H R-C-COO- -N: H-I-H H Aminoacid O:C-H I -ol4-'rcH2o-@ ttl H3Ci-2 I H Pyridoralphosphate Glucose t J Fiq.7.24 : Pyridoxalphosphate(PLP)integratesamino acid and carbohydrate metabolisms (Alanine and aspartatearc the amino acids respectively converted to pyruvate and oxaloacetate, the keto acids). R-C-COO- tl o o-Keto acid Schifl base Fig.7.25 : Formationof Schiff basein transamination'
  • chapter 7 : VITAMINS 745 FiS. l.?6 | Roteofpyridoxinein tryptophan metabolism(pLp-pyridoxalphosphate). Tyrosine--+ DOPA Dopamine___+ coz Norepinephrine__+Epinephrine 3. Pyridoxal phosphate is required for the synthesisof &amino levulinic acid, theprecursor for heme synthesis. Glycine 6-Amino-....|Heme SuccinylCoA levulinic acid(ALA) excretion of xanthurenate in urine is an indicationof 86 deficiency. 5. PLPplaysan importantrole in the metabo_ lism of sulfurcontainingamino acids(Fig.7.27). Transsulfuration(transferof sulfur) from homo_ cysteine to serine occurs in the synthesis 6. Deaminationof hydroxylgroupcontaining amino acidsrequirespLp. Serine Pyruvate+ NH3 Tryptophan iY 3-Hydrorykynurenine NAD+ NADP+ Threonine o-Ketobutyrate+ NHg . 7. Serineis synthesizedfrom glycineby a plp dependentenzyme hydroxymethyltr"nsfer"ru. 9. PLPis neededfor the absorptionof amino acids from the intestine. i 0. Adequateintakeof 86 is usefulto prevent hyperoxaluriaand urinarystoneformation. Reconnmended dietary allowance (RDAI Dietary sources Animal sourcessuch as egg yolk, fish, milk, meatare rich in 86. Wheat,corn, cabbage,roots and tubersare good vegetablesources. Methionine---+ Homocysterne Cystathionine Fiq.7.27 : Role of pyridoxinein the metabolismof sy anino acids (pLp-pyridoxat pnospniti).-
  • 146 BIC]CHEMISTFIY t-,;;;j i,."ian81, $rf?r!Storrrs Pyridoxine deficiency is associated with neurological symptoms such as depression, irritabilitv,nervousnessand mental confusion. Convulsions and peripheral neuropathy are observedin severedeficiency.Thesesymptoms are related to the decreased svnthesis of biogenic amines (serotonin, CABA, norepinephrineand epinephrine).ln children,86 deficiency with a drasticallyreduced CABA productionresultsin convulsions(epilepsy). Decreasein hemoglobin levels, associated with hypochromicmicrocyticanaemia,is seen in 86 deficieny.This is due to a reduction in heme production. The synthesisof niacin coenzymes(NAD+ and NADP+) from tryptophan is impaired. Xanthurenic acid, produced in high quantitiesis excreted in urine, which serves as a reliable index (particularlyaftertryptophanload test)for 86 deficiency. Dietarydeficiencyof pyridoxineis ratherrare and is mostly observedin women taking oral contraceptives,alcoholicsand infants. *i *i..rlr-rtirr:ils F,i deficienerr Isoniazid(isonicotinicacid hydrazide,INH) is a drug frequently used for the treatment of tuherculosis. lt combines with pyridoxal phosphateto form inactivehydrazonederivatives which inhibit PLP dependent enzymes. Tuberculosis patients, on long term use of isoniaTid,developperipheralneuropathywhich respondsto B6 therapy. The drug penicillamine (B-dimethylcysteine) is used in the treatment of oatients with rheumatoid arthritis, Wilson's disease and cystinuria.Thisdrug alsoreactswith PLPto form inactivethiazolidinederivative. Administrationof drugsnamelyisoniazidand penicillamineshouldbe accompaniedby pyrido- xine supplementationto avoid 86 deficiency. + ' :-r'lil,:r:r1n€iEiltaggniSts lsoniazid, deoxypyridoxine and methoxy pyridoxineare the antagonistsof vitamin 86. Toxic effects of overdose vitamiil B6 Excessuse of vitamin 86 Q.5 g/day) in the women of premenstrualsyndromeis associated with sensory neuropathy.Some workers have suggestedthat vitamin 86 morethan 200 mg/day may causeneurologicaldamage. Biotin (formerly known as anti-egg white injuryfactor,vitamin87 or vitaminH) is a sulfur containingB-complexvitamin.lt directlypartici- pates as a coenzyme in the carboxylation reactions. Boas (l 927) observed that rats fed huge quantityof raw egg white developeddermatitis and nervousmanifestations,besidesretardation in growth. She however, found that feeding cooked egg did not produce any of these symptoms.lt was latershownthat the eggwhite injuryin ratsand chickswasdueto the presence of an anti-vitaminin egg white. The egg-white injury factor was identified as a glycoprotein- avidin and biotin was called as anti-egg white injury factor. Shemistry Biotin is a heterocyclicsulfur containing monocarboxylicacid. The structureis formed by fusion of imidazole and thiophene rings with a valeric acid side chain (Fig.7.2A. Biotin is covalentlybound to e-aminogroup of lysineto form biocytin in the enzymes.Biocytin nray be regardedas the coenzyme of biotin. Sitefor Fiq.7.28 : Structureof biotin withbinding sites.
  • Chapter 7 : VITAMINS 147 Biochernical functions Biotin serves as a carrier of COz in carboxylation reactions. The reaction cataiysed by pyruvate carboxylase, converting pyruvate to oxaloacetatehasbeeninvestigatedin detail.This enzyme has biotin bound to the apoenzyme linked to the e-amino group of lysine,forming the activeenzyme(holoenzyme).Biotin-enzyme reactswith CO2 in presenceof ATP (provides energy) to form a carboxybiotin-enzyme complex.This high energycomplexhandsover the CO2 to pyruvate(carboxylationreaction) to produceoxaloacetate(Fig.7.20. As a coenzyme,biotin is involved in various metabolicreactions. 1. Gluconeogenesisand citric acid cycle : The conversion of pyruvate to oxaloacetate by biotin dependent pyruvate carboxylase (describedabove)is essentialfor the synthesisof glucose from many non-carbohydratesources. Oxaloacetateso formed is also requiredfor the continuousoperationof citric acid cycle. 2. tatty acid synthesis : Acetyl CoA is the startingmaterialfor the synthesisof fatty acids. The very first step in fatty acid synthesisis a carboxylationreaction. AcetylCoA Biotin MalonylCoA Acetyl CoAcarboxylase 3. Propionyl CoA is produced in the meta- bolism of certain amino acids (valine,isoleu- cine, threonine etc.) and degradationof odd chain fatty acids. lts further metabolism is dependenton biotin. PropionyrcoA Bioti:.-- ..-- ) PropionylCoAqafbq,rylase MethylmalonylCoA 4. ln the metabolismof leucine,the following reactionis dependenton biotin. B-MetlrylcrotonylCoA B-MethylglutaconylCoA lNote : lt was once believed that all the carboxylationreactionsin the biologicalsystem are dependenton biotin. This was later proved to be wrong. There are a few carboxylation reactions which do not require biotin e.g. formationof carbamoylphosphatein ureacycle, incorporationof CO2 in purine synthesis.l Recosnmended dietary allowance (RDAI A daily intake of about 100-300 mg is recommended for adults. In fact, biotin is normally synthesizedby the intestinalbacteria. However, to what extent the synthesizedbiotin contributes to the body requirementsis not clearlvknown. Metary sources Biotiniswidelydistributedin bothanimaland plant foods. The rich sourcesare liver, kidney, egg yolk, milk, tomatoes,grainsetc. Deficiency synrptonrs The symptomsof biotin deficiency include anemia, loss of appetite, nausea, dermatitis, 0 il Blotin-Enz Carboxybiotln-enzymecomplex o tl cH3-c-cocr Pyruvate U tl*s-c-cH2-c-coo- Oxaloacetate Fig.7.29: Roleofbiotinin thecatuorylationreaction, z) catalysed by the enzyme pyruvate cafuorylase (Enz-Enzyme).
  • 148 BIOCHEMISTRY glossitisetc. Biotin deficiency may also result in depression, hallucinations, musclepain and dermatitis, Biotin deficiency is uncomrnon, since it is well distributedin foods and also supplied by the intestinalbacteria.The deficiency may however, be associated with the following two causes. 1. Destructionof intestinalflora due to prolonged use of drugs such as sulfonamides. 2. High consumptionof raw eggs.The raw egg white contains a glycoprotein- avidin, which tightly binds with biotin and blocks its absorption from the intestine.An intakeof about20 raw eggs per day is needed to produce biotin deficiency symptoms in humans. Consumptionof an occasionalraw egg will not resultin deficiencv. Antagonists Desthiobiotin,biotin sulphonic acid are biotin antagonists. Pantothenic acid (Creek : pantos- everywhere),formerly known as chick anti-dermatitisfactor (or filtratefactor)is widely distributed in nature. lt's metabolic role as coenzyme A is also widespread. Chemistry and synthesis of coenzyme A Pantothenic acid consists of two components,pantoicacid and p-alanine, held togetherby a peptidelinkage.Synthesisof coenzymeA from pantothenateoccursin a series of reactions (Fi9,7.30). Pantothenateis first phosphorylatedto which cysteine is added. Decarboxylation,followed by addition of AMP moietyand a phosphate(eachfrom ATP)results in coenzyme A. The structureof coenzyme A Pantothenlcacld l'^rP l'root + 4'-Phosphopantothonate Cvsteine-.LzATP l)noPv 4'-Phosphopantothenylcysteine +'-Phosphoiantetheine lrlr:P t IYPPi + Dephospho-coenzymeA I J CoenrymeA OH ilt l*-3-"*2-cH2-sH I o- CoenrymeA Fiq.7.30: (A)Summaryol thesynthesisofcoenzymeA frompantothenicacid(B)Structureof coenzymeA. consists of pantothenic acid joined to p-mercaptoethanolamine(thioethanolamine)at oneend.On theotherside,pantothenicacidis heldby a phosphatebridgeto adenylicacid.The adenylicacid is madeup of adenine,and a phosphatelinkedto carbon-3of ribose.
  • Chapter 7 : VITAMINS 149 O O CHr-C-S-CoA ilil| R-C-S-CoA H3C-C-S-CoA CH2-COOH Acyl CoA AcetylCoA SuccinylCoA Fig.7.31 : Selectedexamplesof compounds bound to coenzymeA. Biochemical functions The functionsof pantothenicacid are exerted through coenzyme A or CoA (A for acetylation). CoenzymeA is a central molecule involved in all the metabolisms(carbohydrate,lipid and protein).lt plays a unique role in integrating various metabolic pathways. More than 70 enzymes that depend on coenzyme A are known. CoenzymeA hasa terminalthiol or sulfhydryl group (-SH) which is the reactivesite, hence CoA-SH is also used. Acyl groups (free fatty acids)are linked to coenzymeA by a thioester bond, to give acyl CoA. When bound to acetyl unit, it is called acetyl CoA. With succinate, succinyl CoA is formed. There are many other compoundsbound to coenzymeA. Coenzyme A servesas a carrier of activated acetyl or acyl groups (as thiol esters).This is comparable with ATP which is a carrier of activatedphosphorylgroups. A few examples of enzymes involved the participationof coenzymeA are given below. Pyruvate Acetyl CoA Pyruvatedehydrogenase o-Ketoglutarate c.Ketoglutaratedehydr€€nase SuccinylCoA Fattyacid AcylCoA ThioHnass In some of the metabolic reactions,group transferis importantwhich occursIn a coenzyme A bound form. AcetylCoA+ Choline-----+Acetylcholine+ CoA AcetylCoA+ Oxaloacetat€---) Citrate+ CoA SuccinylCoA+ Acetoacetate-----+AcetoacetylCoA + Succinate CoenzymeA may be regardedas a coenzyme of metabolic integration, since acetyl CoA is a central molecule for a wide variety of biochemicalreactions,as illustratedin Fig.7.32. Succinyl CoA is also involved in many reactions,including the synthesisof porphyrins of heme. Besides the various functions through coenzyme A, pantothenic acid itself is a component of fatty acid synthase complex and is involved in the formationof fatty acids. Recommended dietary allowance (RDAI The requirement of pantothenic acid for humansis not clearlyknown. A daily intakeof about 5-10 mg is advisedfor adults. Dietary sources Pantothenicacid is one of the most widely distributedvitaminsfound in plantsand animals. The rich sourcesare egg,liver,meat,yeast,milk etc. Deficiency symptoms It is a surpriseto biochemiststhat despitethe involvementof pantothenicacid (ascoenzymeA) in a great number of metabolic reactions,its Carbohydrates Aminoacids Faftyacids o tl TCA cycle I+ Energy FatV acids I+ Triacyl- glycerols Ketone bodies I+ Energy Detoxi- ficationCholesterol I VitaminD, steroidhormones Fiq.7.32 : An overviewof formationand
  • 150 BIOCHEMISTFIY deficiency manifestationshave not been reportedin humans.This may be due to the widespread distributionof this vitamin or the symptomsof pantothenicacid may be similar to other vitamin deficiencies.Dr. Copalan,a world renowned nutritionist from lndia, linked the burning feet syndrome (pain and numbnessin the toes, sleeplessness,fatigue etc.) with pantothenicacid deficiency. Pantothenicacid deficiency in experimental animals results in anemia, fatty liver, decreased steroidsynthesisetc. Folic acid or folacin (Latin : folium-leaf) is abundantly found in green leafy vegetables. lt is important for one carbon metabolism and is required for the synthesisof certain amino acids, purinesand the pyrimidine-thymine. coo- Glutamate OH 8.tl-9t-"oo i cHo ;l i 9Hz Paraamino benzoicacid Pteroicacid 5,6,7,8-Tetrahydrofolic acid coo- Chennistry Folic acid consists of three components- pteridinering,p-aminobenzoicacid (PABA)and glutamicacid (1 to 7 residues).Folicacid mostly hasone glutamicacid residueand is known as pteroyl-glutamic acid (PGA). The active form of folic acid is tetrahydrofolate (THF or FHl. lt is synthesized from folic acid by the enzyme dihydrofolate reductase. The reducing equivalents are providedby 2 molesof NADPH. The hydrogen atomsare presentat positions5,6,7 and 8 of THF (Fig.7.33). Absarption, transport and storage Most of the dietary folic acid found as polyglutamatewith 3-7 glutamateresidues(held by peptide bonds) is not absorbed in the Ftq.7.33: Conversionof folicacidto tetrahydrofolicacid(THF). intestine.The enzyme folate conjugasepresent in duodenumand jejunum splitsthe glutamate residues.Only the monoglutamateof folic acid is absorbedfrom the intestine.However, inside the cells, tetrahydrofolates are found as polyglutamates(with 5-6 amino acid residues) derivatives,which appearto be biologicallymost potent. As polyglutamate, folic acid is stored to some extent in the liver. The body can store 10-12 mg of folic acid that will usuallylastfor 2-3 months. In the circulation, Ns-methyl tetrahydrofolateis abundantlypresent. Biochenrical functions Tetrahydrofolate(THFor FHa),the coenzyme of folic acid, is actively involved in the one carbon metabolism. THF servesas an acceptor or donor of one carbon units (formyl, methyl etc.) in a variety of reactionsinvolving amino acid and nucleotidemetabolism.
  • Chapter 7 : VITAMINS 151 The one carbon units bind with THF at positionys 61 y10 or on both Ns and Nlo of pteroyl structure. The attachment of formyl (-CHO) at position 5 of THF gives Ns-formyl tetrahydrofolatewhich is commonly known as folinic acid or citrovorum factor. The other commonly found one carbon moietiesand their binding with THF are given below. H CH-(Glu)n THF-I carbon derivative N5-FormylTHF N1o-FormylTHF N5-FormiminoTHF N5,N1o-MethenylTHF N5,Nlo-MethyleneTHF N5- MethylTHF Rgroup(onecarbonunit) -cHo -cHo -CH=NH :CHz -CHs The essentialfunctionsof THF in one carbon metabofism are summarized in Fig.7.34. The interrelationshipbetween the various 1-carbon THF derivatives along with their involvement in the synthesis of different compounds is given in Fig.l5.32 (Chapter 1fl. Many importantcompoundsare synthesizedin one carbon metabolism. 1. Purines(carbon2, 8) which are incorpora- ted into DNA and RNA. 2. Pyrimidine nucleotide-deoxythymidylic acid (dTMP),involved in the synthesisof DNA. 3. Clycine,serine,ethanolamineand choline are produced. 4. N-Formylmethionine,the initiator of protein biosynthesisis formed. Tetrahydrofolate is mostly trapped as Ns-methylTHF in which form it is presentin the circulation. Vitamin Btz is needed for the conversion of Ns-methyl THF to THF, in a reactionwherein homocysteineis convertedto methionine. This step is essential for the liberationof freeTHF and for its repeatedusein one carbon metabolism. In Btz deficiency, conversionof Ns-methylTHF to THF is blocked (more detailsgiven under vitamin 812). Reeomnrenried die*ary allowance {R$A} The daily requirementof folic acid is around 20O 1tg. In the women, higher intakes are recommendedduring pregnancy @0O p{day) and lactation (300 pglday). Dietary s$snrrees Folic acid is widely distriburedin nature.The rich sources are green leafy vegetables,whole grains, cereals, liver, kidney, yeast and eggs. Milk is rathera poor sourceof folic acid. Deficfi*mey syrynptoms Folic acid deficiency is probably the most common vitamin deficiency, observed primariIy in the pregnant women, in both developed (including USA) and developing countries (includingIndia).The pregnantwomen, lactating women/ women on oral contraceptives,and alcoholics are also susceptible to folate Glycine,serine Onecarbon(1C) histidineetc. donors I Y One carbon(1C)moiety 1C_THF Aminoacids purines (glycine,serine)(2,8 carbons) Choline Fiq.7.34 : An overviewof one carbonmetabolism Onecarbonmoiety(1C) acceptedforthesynthesisof Purines Thymidylate (THF-Tetrahydrofolate).
  • 152 BIOCHEMISTFIY deficiency.Thefolic acid deficiencymay be due to (one or more causes) inadequate dietary intake, defective absorption, use of anticonvulsant drugs (phenobarbitone, diIantin, phenyltoin), and increaseddemand. In folic acid deficiency,decreasedproduction of purinesand dTMP is observedwhich impairs reductaseand block the formationof THF. The biosynthesisof purines,thymine nucleotidesand hence DNA is impaired. This resultsin the blockage of cell proliferation. Aminopterin and methotrexate are successfully used in the treatment of many cancers,including leukemia. Trimethoprim (a component of the drug septran or bactrim) and pyrimethamine (antimalarialdrug)arestructurallyrelatedto folic acid. They inhibit dihydrofolatereductase,and the formationof THF. Sulfonamides:Folic acid, as such, cannot enter bacterial cells. However, bacteria can synthesizefolic acid from pteridine,PABA ano glutamate.Sulfonamidesarestructural analogues of PABA.They competitivelyinhibit the enzyme (dihydropteroatesynthase)responsiblefor the incorporationof PABA into pteridineto produce folic acid. Forthis reason,sulfonamidesare used as antibacterialdrugs. Sulfonamides,have no effect on human body, since folic acid is not synthesizedand suppliedthrough the diet. Vitamin 812is also known as anti-pernicious anemia vitamin. lt is a unique vitamin, synthesizedby only microorganismsand not by animalsand plants.lt was the lastvitamin to be discovered. Ghemistry Vitamin Btz is the only vitamin with a complex structure. The empirical formula of vitamin B12 (cyanocobalamin)is C63H9sN1a OlaPCo.The structureof vitamin B12consistsof a corrin ring with a central cohalt afom. The corrin ring is almostsimilarto the tetrapyrrole ring structure found in other porphyrin compoundse.g.heme(with Fe)and chlorophyll (with Mg). The corrin ring hasfour pyrroleunits,just like a porphyrin.Two of the pyrroleunits(A and Dt are directly bound to each other whereasthe othertwo (Band C) are held by methenebridges The Broups namely methyl, acetamide and DNA synthesis. Due to synthesis,the maturation block in DNA erythrocytesis slowed down leading to macrocytic RBC. The rapidly dividing cells of bone marrow are seriously affected. The macrocytic anemia (abnormally large RBC) associated with megaloblasticchanges in bone marrow is a characteristicfeature of folate deficiency. Folicacid deficiencyin pregnantwomen may cause neural defects in the fetus. Hence high doses of folic acid are recommended in pregnancyto preventbirth defects,. Folic acid is associatedwith the metabolism of histidine. Formiminoglutamate(FICLU), formed in histidinemetabolismtransfersthe one carbon fragment,formimino group (-CH=NH) to tetrahydrofolateto produce Ns-formimino THF. In case of folic acid deficiency, FIGLU accumulatesand is excretedin urine. Histidine loadtestutilizingthe excretionof FIGLUin urine is used to assessfolic acid deficiencv. Folic acid and hyperhomocystelnemia Elevatedplasma levels of homocysteineare associatedwith increasedrisk of atherosclerosis, thrombosis and hypertension. Hyperhomo- cysteinemiais mostly due to functional folate deficiencycausedby impairmentto form methyl- tetrahydrofolate by the enzyme methylene tetrahydrofofate reductase (See Fig.7.39. This resultsin a failure to convert homocysteineto methionine.Folic acid supplementationreduces hyperhomocysteinemia,and therebythe risk for varioushealthcomplications. Folic acid antagonists Aminopterinand amethopterin(alsocalled as methotrexate)are structuralanaloguesof folic acid. They competitively inhibit dihydrofolate a of
  • Ghapter 7 : VITAMINS 153 A I lle. -N r-{ -N N-< Flg. 7.35: Structureof vitamin8,, (cyanocobalamin). propionamidearethe substituentson the pyrrole rings. Vitamin Btz has cobalt atom in a coordinationstateof six. Cobalt presentat the centre of the corrin ring is bonded to the four pyrrole nitrogens.Cobalt also holds (below the corrin plane) dimethylbenzimidazole (DMB) containing ribose 5-phosphate and amino- isopropanol. A nitrogen atom of dimethyl- benzimidazoleis linked to cobalt. The amide group of aminoisopropanolbindswith D ring of corrin. The cobalt atom also oossessesa sixth substituentgroup located above the plane of corrin ring (Fig.7.35).The substituentgroup may be one of the following 1. Cyanide(predominant)in cyanocobalamin (Brz") Hydroxyl in hydroxycobalamin(B126) Nitritein nitrocobalamin(8126). Therearetwo coenzymeformsof vitamin 812 (Fig.7.36). (a) SlDeoxyadenosylcobalamin,cyanide is replaced by 5' deoxyadenosineforming an unusalcarbon cobalt bond. (b) Methylcobalaminin which cyanide is replacedby methyl group. Absorption, transport and storage The vitamin B12 is presentin the diet in a bound form to proteins.B12is liberatedby the enzymes (acid hydrolases)in the stomach.The dietary sourceof B12is known as extrinsicfactor of Castle.The stomachsecretesa specialprotein called intrinsic factor (lF). lt is a glycoprotein (8-15% carbohydrate)with a molecularweight illeffiylcobalamin S'-Deoxyadenosyl- cobalamin CHs CHz Ho[]o c 2. 3. Fiq.7.36 : Coenzymederivativesof vitamin8., (Note: Corrin ring represented diagtammatically is identical in all; DMB-Dimethylbenzimidazole).
  • 154 B]OCHEMISTRY DeoxyadenosylBj2 Fig. 7.37: Absorption,transpoftandstorageof vitaminBr, (lF-lntrinsicfactor;TC-Transcobalamins(TC-|, TC-ll). around 50,000. Intrinsic factor is resistantto proteolyticdigestiveenzymes.lF generallyforms a dimer, binds stronglywith 1 or 2 moles of vitamin 812.This binding protectsvitamin 812 againstits uptakeand use by bacteria. The cobalamin-lF complex travels through the gut. The complex bindsto specificreceptors on the surfaceof the mucosalcellsof the ileum. The bindingof the complexand entryof 812into the mucosalcells is mediatedby Ca2* ions. In the mucosal cells, Btz is converted to methyfcobalamin (Fig.7.SV.lt is then transported in the circulation in a bound form to proteins namely transcobalamins (TC-|, TC-ll). Methylcobalaminis mostlybound to TC-l (90%) and to a lesserdegree to TC-ll (10%). lt is believed that TC-l acts as a repository of 872, whife TC-ll mediatesthe tissueuptake of 812. Methylcobalaminwhich is in excessis taken up by the liver,convertedto deoxyadenosyl812and stored in this form. lt is believedthat liver can storeabout 4-5 mg, an amount sufficientto meet the body requirementsof 812for 4-6 years. Biochemical functions About ten enzymes requiring vitamin B12 have been identified.Most of them are found in bacteria (glutamate mutase, ribonucleotide reductaseetc.).There are only two reactionsin mammalsthat are dependenton vitamin 812. 1. Synthesis of methionine from homo- cysteine : Vitamin 812, ds methylcobalaminis used in this reaction. This is an important reaction involving Ns-methyl tetrahydrofolate from which tetrahydrofolate is Iiberated (enzyme-homocysteine methyltransferase or
  • C-aoter 7 : VITAMINS 155 npthionine synthase). This metabolic step ; Enifiesthe interrelationbetween vitamin 812 -'.d folic acid (detailsgiven later) N5.M r:nocystein Methionine 2. lsomerization of methymalonyl CoA to rrccinyl CoA : The degradationof odd chain ;att)' acids, certain amino acids (valine, r=oleucineetc.) and pyrimidines(thymineand -racil) producedirectlyor throughthe mediation of propionyl CoA, an important compound nethylmalonyl CoA. This is converted by the enzyme methylmalonyl CoA mutaseto succinyl CoA in the presence of B.tz coenzyme, deoxyadenosyl cobalamin (Fig.7s$. This .eaction involves hydrogen transfer and intramolecularrearrangement.In 812deficiency, methylmalonylCoA accumulatesand is excreted in urine as methylmalonicacid. Recommended dietary allowance {nOAl A daily intakeof about 3 pg of vitamin B12is adequateto meet the adult requirements.For children, 0.5-1.5 ttg/day is recommended. Duringpregnancyand lactation,the requirement is 4 1tg/day. Dietary sources Foodsof animal origin are the only sources for vitamin B12. The rich sources are liver, kidney,milk, curd,eggs,fish,pork and chicken. Curd is a better sourcethan milk, due to the synthesisoI 812 by Lactobacillus. Vitamin 812 is synthesized only by micro- organisms (anaerobic bacteria). Plants cannot synthesize,hence 812 is never found in plant foods.Animalsobtain 812eitherby eatingfoods, derivedfrom otheranimalsor from the intestinal bacterialsvnthesis. Deileierrcy syrnptoms The most important diseaseassociatedwith vitamin812deficiencyis perniciousanemia.lt is Thymine,uracil tl C-S-CoA H.rC-C-H I coo- MethylmalonylCoA unne o tl C-S-CoA I QHzI r^U Y"i coo- SuccinylCoA YV Citricacidcycle Porphyrins Oddchain fatty acids I Aminoacids (Val,Ile,Thr,Met) I I Fig. 7.38: Roleof vitaminB, in isomerization ofmethylmalonylCoAtosuccinylCoA (t-Blockade in 8,, deficiency). characterized by low hemoglobin levels, decreased number of erythrocytes and neurologicalmanifestations.One or more of the following causesareattributedto the occurrence of perniciousanemia. 1. Autoimmunedestructionof gastricparietal cells that secreteintrinsicfactor. In the absence of lF, vitamin 812cannotbe absorbed. 2. Hereditarymalabsorptionof vitamin B1r. 3. Partialor total gastrectomy-theseindivi- duals become intrinsicfactor deficient. 4. Insufficientproductionof lF and/orgastric HCl, occasionallyseenin older people. 5. Dietary deficiencyof 81, is seen among the strict vegetariansof low socioeconomic group in the developingcountries(lndia,Srilanka etc.).
  • 1' 156 BIOCHEMISTRY Fromthe foregoingdiscussion,it is clear that pernicious anemia is more a diseaseof the stomachthandueto thedeficiencyof vitaminB12. Btz deficiency is also associated with neuronal degeneration and demyelination of nervous system. The symptoms include paresthesia(numbnessand tingling) of fingers and toes. In advancedstages,confusion,lossof memory and even psychosismay be observed. The neurologicalsymptomsof perniciousanemia are believedto be due to the accumulationof methylmalonyl CoA that interferesin myelin sheathformation in two possibleways. 1. The biosynthesisof fattyacids,requiredfor myelin formation, is imparied.This is because, methylmalonyl CoA acts as a competitive inhibitor of malonyl CoA in fatty acid synthesis. 2. Methylmalonyl CoA cari substitute malonyl CoA in fatty acid synthesis,resultingin a new type of branchedchain fatty acids.These fatty acids will disrupt the normal membrane structure. The excretion of methylmalonic acid (elevated)in urine and estimationof serum 812 level are used to assess812 deficiency. Treatment Vitamin 812 is administeredin therapeutic doses('100-1000pg) intramuscularly.Folicacid administrationcan also reversehematological abnormalities observed in Btz deficiency. However, the neurological symptoms persist. Therefore,a combined supplementationof B12 and folate is employedto treatthe patientswith megaloblasticanemias. INTERRELATIONBETWEENFOLIC AC|D AND VITAM|N Br2 -FOLATE TRAP OR METHYL TRAP HYPOTHESIS The deficiencyof eitherfolateor vitamin 812 resultsin a similartype of anemia.Thissuggests a probable biochemical interrelationbetween thesetwo vitamins.Thereis only one metabolic reactionknown, common to folate and vitamin 812 Gig.7.39. In vitamin B12 deficiency, increasedfolate levelsareobservedin plasma.The activityof the enzyme homocysteine methyltransferase (methionine synthase)is low in 812deficiency. As a result, the only major pathway for the conversionof Ns-methylTHF to tetrahydrofolate is blocked and body THF pool is reduced. Essentiallv. almost the entire bodv folate becomes trapped as Ns-methyl THF. This is known as folate trap or methyl frap. ln this manner, B12 deficiency results in decreased folate coenzymes that leads to reduced nucleotideand DNA svnthesis. Althoughthe tissuefolate levelsare adequate or high, there is a functional folate deficiency due to the lack of THF pool. The outcomeis the developmentof megaloblasticanemia.Adminis- trationof the amino acid methioninehas been shown to partiallycorrectthe symptomsof 812 deficiency. This is due to the fact that the formation of N5-methylTHF is inhibited by S-adenosylmethionine.A combined therapy of vitamin812and folic acid is generallyemployed to treat the patientswith megaloblasticanemia. Besidesthe vitamins describedabove, there are many other compoundspresentin foods as accessoryfactors.Earlierworkershavedescribed thesefactorssometimeor the other,as essential to higher animals. However, their essential natureand requirementin humanshasnot been established.Although not essentialin the diet, they perform many importantfunctions in the body. Selected examples of such substances which may be regarded as vitamin like compoundsare describedhere. Choline is trimethylhydroxyethanolamine. H3 H3C- +-CH2-CH2OH cHs
  • Chapter 7 : VITAMINS 157 One carbon / / Metylcoba-/ HomocYsteine lamrn Fiq.7.39 : lnterrelationshipbetuveenfolicacid and vitaminB,r. It can be synthesizedin the body (from serine).lt is also availablefrom many dietary sources(e.g.milk, eggs,liver, cerealsetc.). 6 icchenrica! functlons 1. Choline, as a component of plrospholipids Llecithins),is involved in membranestructure and lipid transport. 2. Choline preventsthe accumulationof fat in liver (as lipotropic factor). lt promotes the svnthesisof phospholipidsand lipoproteinsand the disposalof triacylglycerols from liver. 3. Due to the presence of three methyl groups (one carbon fragments), choline is actively involved in one carbon metabolism. 4. Choline is a precursorfor the synthesisof acetylcholinewhich is requiredfor transmission of nerveimoulse. Gholine-an essential nutrient? As such, choline can be synthesizedand reutilizedin humans.This may however, be insufficient to meet the body needs. Some s orkers label choline as an essentialdietarv rutrient with RDA in the range of 400-500 mglday. lnositol is hexahydroxy-cyclohexane.lt is also f,non,nas myo-inositolor meso-inositol. OH Biochemical functions 1. Inositol is required for the synthesisof phosphatidylinosifol (lipositol) which is a cons- tituent of cell membrane. 2. ft acts as a lipotropic factor (along with choline)and preventsthe accumulationof fat in liver. 3. For some hormones, inositol acts as a secondmessengerat the membranelevel for the releaseof Ca2+ions. 4. lnositolconcentrationin the heartmuscle in high, the significanceof which however, is not known. 5. Phytin is hexaphosphateof inositolfound is plants.lt preventsthe absorptionof iron and calcium from the intestine. Lipoic acid (thioctic acid) is a sulfur containingfattyacid (6,8-dithiooctanoicacid).lt existsin an oxidized and reducedform. Lipoic acid is fat as well as water soluble.
  • 158 BIOCHEMISTRY Frrg-Crir-cH-(cH2)4cooH l-lrFl-lr-cH-{cH2)1--{ooH llll s--s sF sH Llpolc acld Lipolc acld (oxidized) (reduced) Lipoicacid is involvedin the decarboxylation reactionsalong with other vitamins(thiamine, niacin, riboflavin and pantothenicacid). The conversion of pyruvate to acetyl CoA (by pyruvate dehydrogenase)and a-ketoglutarateto succinylCoA (by cx,-ketoglutaratedehydrogenase) requireslipoic acid. In recentyears,administrationof high doses (100-600 mg/day) of lipoic acid (or dihydro- lipoic acid)is gainingimportance.Beingfat and water soluble,it can comfortablyreachvarious tissues.Thetherapeuticapplicationsof lipoicacid are relatedto itsantioxidantproperty(regardedas universalantioxidant),someof them are listed . Reduces the free radicals in brain that otherwise contribute to Alzheimer's disease and multiplesclerosis. . Lipoic acid stimulates production of glutathione(GSH), besides helping in the recycleof vitaminsE and C. . Reducesinsulinresistance,and bringsdown plasmalow densitylipoproteins. . May be usefulin the preventionof strokeand myocardialinfarction. BIoMEDICAL/ GLINICALCONGEPTS aerDistinct deliciency conditions of certain B-complex uitamins are known Thiamine - Beri-furi Niocin - Pellagra Riboflauin - Cheilosis, glossifis Pyridoxine - Peripheral neuropathy Folic acid - Macroc7ticanemia Cobalomin- Perniciousanemia B-complex uitamin deficienciesare usuolly multiple rather than individuol with ouerlopping symptoms. A combined therapy oJ vitamin Bp and lolic ocid is commonly employed to treat the patients of megaloblosticonemias. Megodosesof niacin are useful in the treatment ol hyperlipidemia. Long term use of isoniazidfor the treatment of tuberculo.siscouses86 deficiency. Folic acid supplementotion reduces eleuated plasmo homocysteine leuel which is associatedwith atherosclerosisand thrombosis. Sulfonamides serue as antibacterial drugs by inhibiting the incorporation of PABA to produce folic ocid. Aminopterin and amethopterin, the structural analoguesof folic acid, are employgd in the treotment of concers. ns' Lipoic ocid is therapeuticolly uset'ulas an antioxidant to preuent stroke, myocordial infarction, etc.
  • Chatrter7 : VITAMINS 159 Paraaminobenzoicacid (PABA)is a structural constituentof folic acid. PABAmay be regarded as a vitamin in another vitamin (folic acid) NHz Sulfonilamide The deficiencyof PABAwas firstfound to be associatedwith failure of lactationand greying of black hair in rats.The specificfunctionsof PABA in humans,exceptthat it is a component of folic acid, have not been identified. PABA is synthesizedby the bacteriaand is essential for their groMh. The sulfa drug sulfonilamide(p-aminobenzenesulfanilamide)is a structuralanalogue of PABA. Sulfonilamide competeswith PABA and actsas a bacteriostatic agent. Ingestion of large doses of PABA will compete with the action of drugs and thereforeshouldbe avoidedduringsulfonilamide therapy(tradename-sulfonamides). Szent-Cyorgi and his associates (1936) observedthat flavonoids, isolatedfrom lemon peel (known as citrin) were responsiblefor maintenanceof normal capillary permeability. The term vitamin P (P for permeability)was used to this group of substances.However, they are commonly known as bioflavonoids. Bioflavonoidsact as antioxidantsand protect ascorbic acid from being destroyed. lt is suggestedthat this antioxidant property may be responsible for maintenance of capillary permeability.Bioflavonoidshave been used to correctthe vascularabnormalityin humans. Bioflavonoidsare found in peel and pulp of citrus fruits, tobacco leaves and many vegetables.The requirementof thesecompounds in humanshas not been established. Antivitamins are antagonisticto (opposeand block) the action of vitamins.They usually have structuralsimilaritieswith vitamins.Administration of antivitaminscausesvitamin deficiencies.The commonantivitaminsarediscussedasantagonists for each vitamin. CCIOH NHz PABA
  • 160 BIOCHEMISTRY ]. 2. 3 4. 5. 6. 7. 9. 10. 11 72. Vitamins qre occessorglood factors required in the diet. Theg are classified os t'ot soluble(A, D, E and K) ond water soluble(B-complexand C). Vitamin A is inuoluedin uision,proper growth, diJJerentiationand maintenonceoJ epithelialcells. lts deficiencyresu/fsin night blindness. The actiueform ol uitaminD is colcitriolwhichfunctions like a steroidhormoneand regulatesplasmaleuelsol calciumond phosphate.VitaminD det'iciencyleadsto rickets in childrenand osteomalaciain adults. VitaminE is a naturalantioxidantnecessar7for normal reproductionin manyanimals. VitaminK hasa specificcoenzymeJunction.lt catalysesthe carboxylationof glutamic acid residuesin blood clotting factors(Il, Vil, IX and X) and conuertsthem to actiue form. Thiamine (Bl), as a cocarboxylase(TPP) is inuolued in energy releasingreactions. Its deficiency leads to beri-beri. The coenzymesof ribot'lauin(FAD and FMN)and niacin (NAD+ and NADP+)take part in a uoriety of oxidation-reductionreactions connected with energg generation. RiboJlauindet'icienc7resultsin cheilosisand glossitiswhereasniacindeficiencqleadsto pellagro. 8 Pyridoxal phosphate (PLP), the coenzyme of uitamin F6, is mostly associatedwith amino acid metoboltsm. PLP participates in transomination, decarboxylotion, deaminotionand condensationreactions. Biotin (antiegg white injury factor) participates as o coenzyme in corboxylation reactionsof gluconeogenesis,fatty acid sgnthesisetc. CoenzymeA (of pantothenic acid)is inuoluedin the metabolismol carbohgdrates,/ipids and amino acids,and their integration. Tetrohydrofolate(THF} the coenzymeof t'olic acid porticipates in the transt'erof one carbon units (formyl, methgl etc.) in amino acid and nucleotide metabolism. Megaloblasticonemia is cousedb9 t'olic acid deficiency. Vitomin Bp has two coenzymes,deoxyadenosylcobalominand methylcobalamin.Bp deficiency resultsin pernicious anemia. 73. Vitamin C (ascorbicacid)is inuoluedin the hydroxylation of proline and lgsinein the formation of collagen. Scuruyis causedby ascorbicacid deficiency.TherapeuticuseoJ megadosesol uitamin C, to cure euerythinglrom common cold to cancer,hasbecome controuersial. 74. Certain uitamin like compounds (choline, inositol, PABA, lipoic acid) participate in many biochemicalreoctions.
  • Chapter 7 : VITAMINS 161 I. Essayquestions 1. Classifyvitaminsand brieflydiscusstheirfunctionsand deficiencydisorders. 2. Describethe chemistry,biochemicalfunctions,daily requirements,sourcesand deficiency manifestationsof vitaminA. 3. Writean accountof folic acid involvementin one carbonmetabolism. 4. Discussthebiochemicalfunctionsof vitaminC.Add a noteon thetherapeuticuseof megadoses of thisvitamin. 5. Write brieflyaboutthe coenzymesinvolvedin oxidation-reductionreactions. II. Short notes (a) Vitamin D is a hormone-justify,(b) Thiaminepyrophosphate,(c) Coenzymesof niacin, (d) Pyridoxalphosphatein transamination,(e) Folatetrap, (f) Tocopherol,(g) Vitamin K in carboxylation,(h) Biocytin,(i) Choline,(j) Pernrcrousanemra. III. Fill in the blanks TheA in coenzymeA standsfor Thevitamincontainingisoalloxazinering The vitaminthat is regardedas a vitaminin searchof a disease Anti-tuberculosisdrug, isonicotinicacid hydrazide(lNH) leads to the deficiencyof vitamin The egg injuryfactorpresentin raw eggwhite The'burningfeetsyndrome'in manisassociatedwith thedeficiencyof. The vitaminthat is synthesizedby only microorganisms ThethreeDs in pellagrastandfor, and The fat solublevitaminrequiredfor carboxylationreaction FICLU (formimino glutamic acid) is excreted in urine in the deficiency of - rlultiple choice questions 'hich one of the vitaminA functionsas a steroidhormone ar Retinal(b) Retinol(c) ProvitaminA (d) F-Carotene. '- Thefunctionallyactiveform of vitaminD is a Cholecalciferol(b) Ergocalciferol(c) Dehydrocholesterol(d) Calcitriol. ' j T^e metaboliteexcretedin urine in thiaminedeficiency a Pvruvate(b)Glucose(c) Xanthurenicacid (d) FICLU. 'r -^e coenzymedirectlyconcernedwith the synthesisof biogenicamines TPP(b) NADP+(c) Biotin(d) Pyridoxalphosphate. c acid antagonist(s)usedin the treatmentof cancer iethotrexate(b)Trimethoprim(c) Sulfonamide(d)All the three. 1. 2. 3. I 6 q ,l ). 6. r itamin I
  • Sl eiotogicalOxidation
  • DigestionandAbsoqption The natutal Joodctu|fs speah: "Complex is the ingestedfood, But digestedto simltler products, Absarbed by intestinal mwcosalcelb, Assimilatedand utilized by ail celk." f ood is the basicand essentialrequirementof I man for his verv existence.The food we eat consists of carbohydrates, proteins, lipids, vitamins and minerals.The bulk of the food ingestedis mostlyin a complexmacromolecular form which cannot,as such,be absorbedby the body. Digestion rb a process involving the hydrolysis of large and complex organic molecules of foodstuffs into smaller and preferably water-soluble molecules which can be easily absorbed by the gastrointestinal tract for utilization by the organism. Digestion of macromoleculesalsopromotesthe absorptionof fat solublevitaminsand certainminerals. Cookingof the food, and mastication(in the mouth) significantlyimprove the digestibilityof foodstuffsby the enzymes. i - f;qi lrsrl*rtlstinal trae t Digestion as well as absorption are complicatedprocessesthat occur in the gastro- intestinaltract(ClT)involvingmanyorgans.The Productionof salivacontainingc-amylase; partialdigestionof polysac'charides Elaborationof gastricjuice with HCI and proteases;partialdigestionof proteins Releaseof NaHCO3and manyenzymes requiredlor intestinaldigestion Synthesisof bileacids Storageol bile Organ Major function(s) Liver Gallbladder Smallintestine Finaldigestionof digestedproducts Largeintestine Mostlyabsorptionof utilizationof certain unabsorbedfoods foodstutfs;absorptionol electrolytes;bacterial non-digestedand/or diagrammaticrepresentationof CIT is depicted in Fig.8.l, and the essential organs with their respectivemajor functions are given in Table 8.1. The digestiveorganspossessa large 165
  • 156 BIOCHEMISTFIY FIg. 8.1 : Diagrammaticrepresentation ot gastrointestinaltract. reservecapacity. For instance,pancreassecretes enzymes 5-10 fold higher than required for digestionof foods normally ingested. The digestion and absorptionof individual foods,namelycarbohydrates,proteins,lipidsand nucleic acids,is describedhere.Thegastrointestinal hormones are discussed under hormones (Chapter l9). The principal dietary carbohydrates are polysaccharides(starch,glycogen),disaccharides (lactose,sucrose)and, to a minor extent,mono- saccharides(glucose,fructose).The structuresof carbohydratesare described in Chapter 2. Digesticn The digestionof carbohydratesoccursbriefly in mouth and largely in the intestine. The polysaccharidesget hydrated during heating which is essentialfor their efficient digestion. The hydrolysisof glycosidicbondsis carriedout by a group of enzymes called glycosidases (Fi9.8.2). These enzymes are specific to the bond, structure and configuration of mono- saccharideunits. Digestion in the mouth : Carbohydratesare the only nutrientsfor which the digestionbegins in the mouth to a significantextent.During the process of mastication, salivary a-amylase (ptyalin) actson starchrandomly and cleavesc- 1,4-glycosidic bonds. The products formed include cr-limit dextrins,(containingabout 8 glucoseunitswith one or more o-1,6-glycosidic bonds)maltotrioseand maltose. Carbohydratesnot digestedin the stomach r The enzyme salivaryamylaseis inactivatedby highacidity(low pH) in the stomach.Consequently, the ongoingdegradationof starchis stopped. Digestionin the small intestine: The acidic dietary contents of the stomach, on reaching small intestine,are neutralizedby bicarbonate produced by pancreas. The pancreatic a-amylase acts on starch and continues the digestionprocess.Amylase specificallyacts on a-l,4-glycosidicbondsand not on q,-1,6-bonds. H"O----l' |Glvcosidase Oesophagus Stomach Gallbladder + Duodenum (- 0.25m) Pyloric sphincter Pancreas +- Jejunum (- 2.3m) {- lleum (- 4.6m) Small intestine Fig. 8.2 : Hydrolysis of a glycosidic bond.
  • Chapten I : DIGESTIONAND ABSOFIPTION 167 a (1-4) Amylopectin lo-nmyase + (l F. ( -)!somaltose <H Maltotriose Fig. 8.3 : Degradation of amylopectin by salivary or pancreatic a-amylase. The resultant products are disaccharides (maltose, isomaltose) and oligosaccharides (Fig.8.3). The final digestion of di- and oligo- saccharides to monosaccharides (Fig.8.4) primarily occurs at the mucosal lining of the upper jejunum. This is carried out by oligosaccharidases (e.g. glucoamylase acting on amylose) and disaccharidases (e.g. maltase, sucrase, lactase).The enzyme sucrase is capable of hydrolysing a large quantity of table sugar (sucrose). In contrast, Iactase (p-galactosidase) is the rate- limiting, and, consequently,the utilizationof milk sugar(lactose)is limited in humans. Absorption of monosaccharides The principal monosaccharidesproduced by the digestion of carbohydratesare glucose, fructose and galactose. Of these, glucose accounts for nearlv 80o/o of the total monosaccharides.The absorption of sugars mostly takes place in the duodenum and upper jejunum of small intestine. Pancreatico-amylase, a-Glucoamylase-------* lsomaltase,Maltase Lactase Sucrase LowpHstops amylaseactivity Fig. 8.4 : Overviewof digestionof carbohydrates.
  • Y' 168 BIOCHEMISTRY There exists a considerable variation in the absorption o'f different monosaccharides. The relative rates of absorption of important monosaccharides in comparisonwith glucoseare given below Capillaries Clucose Calactose Fructose Mannose Xylose Arabinose 100 110 43 20 15 9 It is observed that hexosesare more rapidly absorbed than pentoses. Further, among the monosaccharides,galactoseis most e{ficiently absorbed followed by glucoseandfructose.Insulinhasno effect on the absorption of sugars. lVleeharnisnrr ei!$ahsorption Flg. 8.5 : Transport of glucose across intestinal epithelium (Note : Transport of amino acids also occurs by a similar rehydrationfluid containsglucoseand sodium. Intestinalabsorptionof sodium is facilitatedby the presenceof glucose. The mechanismof absorptionof galactoseis similarto that of glucose.The inhibitorphlorizin blocks the Na+ dependenttransportof glucose and galactose. Absorption of fructose : Fructoseabsorption is relativelysimple. lt does not require energy and is independentof Na+transport.Fructoseis transportedby facilitateddiffusionmediatedby a carrier. Insidethe epithelialcell, most of the fructose is convertedto glucose.The latter then entersthe circulation. Pentosesare absorbedby a processof simple diffusion. $lon"diEestible carbohydrates The plant foods are rich in fibrous material which cannot be digestedeither by the human enzymes or intestinalbacteria.The fibers are chemically complex carbohydrates which includecellulose,hemicellulose,pectins,lignin and gums. Fiber in nutrition is of special impoftancewhich is describedunder nutrition (Chapter 23). Differentsugarspossessdifferentmechanisms for their absorption.Clucose is transportedinto the intestinalmucosalcellsby a carriermediated and energyrequiringprocess(Fig.8.A. Glucose and Na+ share the same transport system(symportwhich is referredto as sodium- dependent glucose transporter.The concen- tration of Na+ is higher in the intestinallumen compared to mucosal cells. Na+, therefore, moves into the cells along its concentration gradient and simultaneously glucose is transported into the intestinal cells. This is mediatedby the samecarriersystem.Thus,Na+ diffusesinto the cell and it dragsglucosealong with it. The intestinal Na+ gradient is the immediateenergy sourcefor glucosetransport. This energyis indirectlysuppliedby ATP since the reentry of Na+ (againstthe concentration gradient)into the intestinallumen is an energy- requiring active process.The enzyme Na+-K+ ATPaseis involved in the transport of Na+ in exchange of K+ against the concentration gradient (for details seeChapter 33). Oral rehydration therapy (ORT) : ORT is the most common treatmentof diarrhea.The oral lntestinalmucosalcell Na+-K+ATPase
  • Ghapter 8: DIGESTIONAND ABSOFIPTION 169 AbnormalitEes of carbohydrate digestion In general,humanspossessan efficientsystem of carbohydratedigestionand absorption.Since only the monosaccharidesare absorbed,any defectin the activitiesof disaccharidasesresultsin the passageof undigesteddisaccharidesinto the largeintestine.Thedisaccharidesdrawwaterfrom the intestinal mucosa by osmosis and cause osmoticdiarrhea.Further,bacterialactionof these undigestedcarbohydratesleadsto flatulence. Disaccharidasesare the intestinal brush borderenzymes.Any alterationin the mucosaof the small intestinecaused by severediarrhea, malnutrition,intestinaldiseasesor drug therapy will lead to a temporary acquired deficiency of disaccharidases.The patientswith such disorders are advised to restrict the consumption of sucroseand lactose. Hereditary disorders with deficiency of individualdisaccharidasesin infantsand children causeintoleranceof specificdisaccharides. Lactose intolerance Defect in the enzyme lactase(F-galactosidase) is the most common disaccharidasedeficiency in humans.lt is estimatedthat more than half of the world's adult population is affected by lactoseintolerance.lt is more commonly found in Africans (blacks)and Asians compared to Europeans.Surprisingly,according to a recent estimate,about 90% of the adult Asians are lactasedeficient.The mechanismof how lactase is lost in adultsis not clear.lt is however,known that there is a reduced production of lactase ratherthan an alterationin enzyme activity. The treatment of lactose intolerance is quite simple. Elimination of lactose from the diet (severe restriction of milk and dairy products) will solvethe problem. Continuedconsumptionof lactoseby lactose intolerant individualscausestypical symptoms of flatulence (describedlater). Sucrase deficiency The deficiencyof the enzyme sucrasecauses intoleranceto dietarysucrose.lt is estimatedthat about 1O"/' of Eskimosof Creenland of North Americans are affected disorder. The treatment is to remove from the diet. and 2"/" by this sucrose The problern of flatulence Flatulence is characterized by increased intestinal motility, cramps and irritation. This occurs after ingestion of certain carbohydrates and is explainedas follows. The carbohydrates(di-, oligo-, and poly- saccharides)not hydrolysedby a,-amylaseand other intestinalenzymes cannot be absorbed. Lactoseis not hydrolysedin some individuals due to the deficiencyof lactase.The di-, and oligosaccharides can be degraded by the bacteriapresentin ileum (lower part of small intestine)to liberatemonosaccharides.The latter can be metabolizedby the bacteria. During the course of utilization of mono- saccharidesby the intestinalbacteria,the gases such as hydrogen, methane and carbon dioxid*-besides lactate and short chain fatty acids-are released. These compounds cause flatulence. The occurrenceof flatulenceafterthe ingestion of leguminous seeds (bengal gram, redgram, beans,peas,soya bean) is very common. They contain severaI nondigestible oligonccharides by humanintestinalenzymes.Thesecompoundsare degraded and utilised by intestinal bacteria causing flatulence. Raffinose containing galactose,glucoseand fructoseis a predominant oligosaccharidefound in leguminousseeds. The proteins subjected to digestion and absorption are obtained from two sources- dietaryand endogenous. The intakeof dietary protein is in the range of 50-100 g/day. About 30-100 e/day of endogenousproteinis derivedform the digestive enzymes and worn out cells of the digestive tract.The digestionand absorptionof proteinsis very efficient in healthyhumans,hencevery little protein(about5-10 S/day)is lost throughfeces.
  • 170 BIOCHEMISTRY Dietary proteinsare denaturedon cooking and therefore,easilydigested. Proteinsaredegradedby a classof enzymes- namely hydrolases-which specifically cleave the peptide bonds, hence known as peptidases. They are divided into two groups 1. Endopeptidases(proteases)which attack the internalpeptide bonds and releasepeptide fragments,e.g. pepsin,trypsin. 2. Exopeptidaseswhich act on the peptide bonds of terminal amino acids. Exopeptidases are subdivided into carboxypeptidases(act on C-terminalamino acid) andaminopepfidases(act on N-terminalamino acid). The proteolyticenzymesresponsiblefor the digestion of proteins are produced by the stomach,the pancreasand the small intestine. Proteinsare not digestedin the mouthdue to the absenceof proteasesin saliva. l. Digestion of proteins by gastric secretion Protein digestion begins in the stomach. Gastric juice produced by stomach contains hydrochloric acid and a protease proenzyme namelypepsinogen. Hydrochloricacid : The pH of the stomachis < 2 due to the presenceof HCl, secretedby parietal(oxyntic)cellsof gastricgland.This acid perfornrstwo importantfunctions-denaturation of proteinsand killing of certainmicroorganisms. The denaturedproteinsare more susceptibleto proteasesfor digestion. Pepsin: Pepsin(Creek: pepsis-digestion)is producedby the serouscells of the stomachas pepsinogen, the inactive zymogen or proenzyme.Pepsinogenis convertedto active pepsin either by autocatalysis,brought about by other pepsin molecules or by gastric HCI (pH < 2). Removalof a fragmentof polypeptide chain (44 amino acids in case of pig enzyme) makesthe inactiveenzymeactiveafterattaining a proper conformation. Pepsin is an acid-stable endopeptidase optimally active at a very low pH (2.0). The activesiteof the enzymecontainstwo carboxyl groups,which are maintainedat low pH. Pepsin Peotides Aminoacids i | , rPe,, I I CCK,secretin Chymotrypsin Elastase Carboxypeptidases (AandB) A isthe mostpredominantgastricproteasewhich preferentiallycleavespeptide bonds formed by amino Broupsof phenylalanineor tyrosineor leucine. Pepsindigestionof proteinsresultsin peptides and a few amino acidswhich act as stimulants for the releaseof the hormone cholecystokinin from the duodenum. Rennin: This enzyme,also called chymosin, is found in the stomachof infantsand children. Rennin is involved in the curdling of milk. lt converts milk protein casein to calcium paracaseinatewhich can be effectivelydigested by pepsin.Renninis absentin adults. ll. Digestion of proteins by pancreatic proteases The proteasesof pancreaticjuice are secreted as zymogens(proenzymes)and then convertedto activeforms. Theseprocessesare initiated by the releaseof two polypeptide hormones,namely cholecystokinin and secretin from the intestine (Fig.8.6). Flg, 8.6 : Formationand activationol Wncreatic
  • Ghapter 8 : DIGESTIONAND ABSOFPTION 171 e@@lll Protein...CO-Nil CH -CO-NH-CH-CO-NH............CO-NH-CH-COO- TI I Enryme PepsinA Trypsin Chymotrypsin Elastase IEnrymeNatureof (ii; Tyr,Phe,Leu Arg,Lys Trp,Tyr,Phe, Leu,Met Ala,Gly,Ser ttatureorG) CarboxypeptidaseA Ala, lle,Leu,Val CarboxypeptidaseB Arg, LYs Fig. 8.7 : Digestion of proteine-Speciticity of enzyme cleavage of peptide bonds. (81 can be from any amino acid) Releaseand activation of zymogens: The key enzyme for activation of zymogen is entero- peptidase(formerly enterokinase)produced by intestinal(mostlyduodenal)mucosalepithelial cells.Enteropeptidasecleavesoff a hexapeptide (6 amino acid fragment)from the N-terminalend of trypsinogento produce trypsin, the active enzyme. Trypsin, in turn, activates other trypsinogenmolecules (autocatalysis).Further, trypsin is the common activator of all other pancreatic zymogens to produce the active proteases,namely chymotrypsin,elastaseand carboxypeptidases(A and B). Specffici$ and action of pancreatic proteases: Trypsin, chymotrypsin and elastase are endopeptidasesactiveat neutralpH. CastricHCI is neutralized by pancreatic NaHCO3 in the intestineand this createsfavourablepH for the action of proteases. The substrate specificity of pancreatic proteasesis depicted in Fi9.8.7. For instance, trypsincleavesthe peptidebonds,the carbonyl (-CO-) group of which is contributed by arginineor lysine. Theamino acid serineisessentialat the active centreto bringaboutthe catalysisof all the three pancreaticproteases,hence theseenzymesare referred to as serine proteases. Action of carboxypeptidases: The pancreatic carboxypeptidases(A and B)aremetalloenzymes that are dependenton Zn2+ for their catalytic activity, hence they are sometimes called Zn-proteases.They also possesscertain degree of substrate specificity in their action. For example, carboxypeptidaseB acts on peptide bondsof COOH-terminalaminoacid,the amino group of which is contributedby arginineor lysine (Fig.8.71. The combinedaction of pancreaticproteases resultsin the formationof free amino acidsand small peptides(2-8 amino acids). lll. Digestion rf proteins by srna!! intestinal enayrnes The luminal surfaceof intestinalepithelial cells contains aminopeptidasesand dipeptidases. Aminopeptidaseis a non-specificexopeptidase which repeatedly cleaves N-terminal amino acids one by one to produce free amino acids and smaller peptides.The dipeptidasesact on different dipeptides to liberate amino acids (Fig.8.8). AhsorptFon nf annlno acads and dipeptides The freeamino acids,dipeptidesand to some extent tripeptides are absorbed by intestinal epithelialcells. The di- and tripeptides,after being absorbed are hydrolysed into free amino acids in the cytosol of epithelial cells. The activities of dipeptidasesare high in these cells. Therefore, after a protein meal, only the free amino acids are found in the portal vein.
  • 172 BIOCHEMISTFIY SmallIntestine Carboxypeptidases Aminopeptidases Dipeptidases I Fig. 8,8 : Overuiew of digestion of proteins. The small intestine possessesan efficient systemto absorbfreeaminoacids.L-Aminoacids are more rapidly absorbedthan D-amino acids. The transportof L-amino acids occurs by an activeprocess(againsta concentrationgradient), in contrastto D-amino acidswhich takesplace by a simplediffusion. Mechanisffi of anrino aeid a$rs*r,gr;ti<rr'r Amino acids are primarily absorbed by a similarmechanism,asdescribedfor the transport of D-glucose.lt is basically a Na+-dependent active processlinked with the transportof Na+. As the Na+ diffuses along the concentration gradient,the amino acid alsoentersthe intestinal cell. BothNa+and aminoacidssharea common carrierand are transportedtogether.The energy is supplied indirectly by ATP (for details, see absorption of monosaccharidesand Fig.8.5). A Na+-independentsystem of amino acid transport across intestinalcells has also been identified.The compound cytochalasinI inhibits Na+-independenttransportsystem. Another transport system to explain the mechanism of amino acid transfer across membranein the intestineand kidnev has been put forth. This is known as y-glutamyl cycle or Meister cycle and involves a tripeptide namely glutathione (y-glutamylcysteinylglycine).Three ATP are utilized for the transportof a single aminoacid by thiscycle.Forthisseason,Meister cycle is not a common transport system for amino acid. However,this cycle is operativefor rapid transportof cysteineand glutamine. The y-glutamylcycle appearsto be important for the metabolism of glutathione,since this tripeptideundergoesrapid turnoverin the cells. Theremay be more physiologicalsignificanceof y-glutamylcycle. AbsorptEon of intact proteins and polypepE!des. Fora shortperiod,immediatelyafterbirth,the small intestine of infants can absorb intact proteinsand polypeptides.The uptakeof proteins occurs by a process known as endocytosisor pinocytosis.The macromoleculesare ingestedby formationof smallvesiclesof plasmamembrane followed by their internalization.The direct absorption of intact proteins is very important for the transferof maternal immunoglobulins (y-globulins)to the offspring. The intact proteinsand polypeptidesare not absorbedby the adult intestine.However, the macromolecular absorption in certain individuals appears to be responsible for antibody formation that often causes food allergy.
  • Chapter 8: DIGESTIONAND ABSORPTION 773 Abnormalities of protein digestion and amino acid absorption Any defectin the pancreaticsecretionimpairs proteinand fat digestion.This causesthe lossof undigestedprotein in the feces along with the abnormal appearanceof lipids. Deficiency of pancreaticsecretionmay be due to pancreatitis (seelater),cystic fibrosisor surgicalremovalof pancreas. Hartnup's disease {neutral amino aciduria} Hartnup is the name of the familv in whom this disease was first discovered. lt is characterized by the inability of intestinal and renal epithelial cells to absorb neutral amino acids. Tryptopftan absorption is most severely affected with a result that typical symptoms of pellagraare observedin the patientsof Hartnup,s disease.This is relatedto the impairmentin the conversionof tryptophanto NAD+ and NADp+, the coenzymesof niacin. There is considerablevariation in the daily consumptionof lipidswhich mostlydependson the economic status and dietary habits. The intakeof fipids is much less(often< 60 {day) in poorersectionsof the society,particularlyin the less developed countries. In the developed countries,an adult ingestsabout 60-150 g of Iipids per day. Of this, more than 90% is fat (triacylglycerol).The rest of the dietary lipid is made up of phospholipids, cholesterol, cholesterylestersand free fattv acids. Lipids are insolubleor sparinglysoluble in aqueous solution. The digestive enzymes, however,are presentin aqueousmedium. This poses certain problems for the digestion and absorptionof lipids. Fortunately,the digestive tract possessesspecializedmachineryto 1. Increasethe surface area of lipids for digestion; 2. Solubilize the digested products for absorption. Minor digestion of liBids in the stomach The digestion of lipids is initiated in the stomach, catalysed by acid-stable lipase. This enzyme(alsocalled linguallipase)is believedto originatefrom the glandsat the back of tongue. Stomachcontainsa separategastric lipasewhich can degradefat containingshortchainfattyacids at neutral pH. The digestion of lipids in the stomachof an adult is almost negligible,since lipids are not emulsified and made readv for lipaseaction.Further,the low pH in the stomach is unfavourablefor the action of gastriclipase. In case of infants,the milk fat (with short chain fatty acids)can be hydrolysedby gastric lipase to some extent. This is because the stomachpH of infantsis closeto neutralitv,ideal for gastriclipaseaction Emulsification of lipids in the small intestine Emulsification is the phenomenon of dispersionof lipids into smallerdropletsdue to reduction in the surface tension. This is accompaniedby increasein the surfaceareaof lipid droplets. Emulsificationis essential for effectivedigestionof lipids, since the enzymes can act only on the surfaceof lipid droplets. More correctly, lipasesact at the interfacialarea betweenthe aqueousand lipid phase. The processof emulsificationoccursbv three complementarymechanisms 1. Detergentaction of bile salts; 2. Surfactantaction of degradedlipids; 3. Mechanicalmixing due to peristalsis. 1. Bile salts : The terms bile saltsand bile acids are often used interchangeably. At physiological pH, the bile acids are mostly presentas anions. Bile salts are the biological detergentssynthesizedfrom cholesterol in the liver. They are secreted with bile into the duodenum.Bilesaltspossesssteroidnucleus,the side chain of which is attachedto either glycine (glycocholic acid) or taurine (taurocholii acid). Forthe synthesisand otherdetailson bile acids, refer cholesterol metabolism (Chapter 14. Bile saltsarethe mosteffectivebiologicalemulsifying
  • a 174 BIOCHEMISTF|Y otl o cH2-o-c-R1 ill R2-c-o-?H ? cH2-o-c-R3 Trlacylglycerol Fig. 8.9 : Enzymatic cleavage of dietary fat. Pan€roariuiDase ? cH2-oH RlcooH R'-C-O-CH + 2Hro - Crr_o, RscooH 2-Monoacylglycerol Freefattyacids agents.They interactwith lipid particlesand the aqueousduodenal contentsand convert them into smaller particles (emulsified droplets). Further,bile saltsstabilizethe smallerparticles by preverrtingthem from coalescing. 2. Surfactantaction of degradedlipids : The initial digestiveproductsof lipids (catalysedby lipase) namely free fatty acids, mono- acylglycerols promote emulsification. These compoundsalongwith phospholipidsare known as surfactants. They are characterized by possessing polar and non-polar groups. Surfactantsget absorbed to the water-lipid interfaces and increase the interfacial area of lipid droplets. Thus the initial action of the enzymelipasehelpsin furtherdigestionof lipids. 3. Besidesthe action of bile saltsand surfac- tants, the mechanical mixing due to peristalsis also helps in the emulsificationof lipids.The smaller lipid emulsion droplets are good substratesfor digestion. DEgestion sf lipids by pancreatic enzyrnes The pancreatic enzymes are primarily responsiblefor the degradationof dietarytriacyl- glycerols,cholesterylestersand phospholipids. Degradation of triacylglycerols (tatl Pancreaticlipase is the major enzyme that digestsdietary fats. This enzyme preferentially cleavesfattyacids(particularlylongchain,above 10 carbons) at position 1 and 3 of triacyl- gfycerofs. The products are 2-monoacylglycerol (formerfy 2-monoglyceride)and free fatty acids (Fi9.8.9).The activity of pancreatic lipase is inhibitedby bile acidswhich are presentalong with the enzvme in the small intestine.This problem is overcomeby a small protein, colipase (mol.wt. 12,000).lt is alsosecretedby pancreas as procolipaseand convertedto active form by trypsin. Colipase binds at the lipid-aqueous interfaceand helpsto anchorand stabilizelipase. Lipid esterase is a less specific enzyme present in pancreatic juice. lt acts on monoacylglycerols,cholesteryl esters,vitamin esters etc. to liberate free fatty acids, The presenceof bile acidsis essentialfor the activity of lipid esterase. Degradation of cholesteryl esters A specificenzymenamelypancreaticcholes- terol esterase (cholesteryl ester hydrolase) cleavescholesterylestersto producecholesterol and free fatty acids (Fig.&JA. Degradation of phospholipids Phospholipasesare enzymes responsiblefor the hydrolysisof phospholipids.Pancreaticjuice is rich in phospholipaseA2 which cleavesthe fatty acid at the 2nd positionof phospholipids. The products are a free fatty acid and a lysophospholipid.Phospholipase42 is secreted as a zymogenwhich is activatedin the intestine by the action of trypsin. An overviewof the digestionof lipidsis given in Fig.8.l | . Absorption of lipids The former and presenttheoriesto explain the absorption of lipids are briefly described hereunder 1. tipolytic theory put forth by Verzar : Accordingto this,fatsarecompletelyhydrolysed to glycerol and free fatty acids. The latter are absorbedeither as soapsor in associationwith bile salts.
  • Chapter a : DIGESTIONAND ABSORPTION 175 --------T--------+ 'Hzo I Cholesterylesterase HO o R-C Cholesterol Fatty acid Cholesterylester Fig. 8.10: Enzymaticcleavageof cholesterylester. 2. Partition theory proposedby Frazer : This theorystatesthatthe digestionof triacylglycerols is partial and not complete. The partially digestedtriacylglycerols,in associationwith bile salts,form emulsions.The lipidsaretakenup by the intestinal mucosal cells. As per this theory, resynthesisof lipidsis not necessaryfor theirentry into the circulation. 3. Bergstromtheory : This is a more recent and comprehensive theory to explain lipid absorption.lt has almost replaced the earlier theories,and is briefly describedhereunder The primaryproductsobtainedfrom the lipid digestionare2-monoacylglycerol,freefattyacids and free cholesterol. Role mf bile salts En llnirl &bs#rpt*{}r! Besidestheir participationin digestion,bile saltsare essentialfor absorptionof lipids. Bile salts form mixed micelles with liDids. These micelles are smaller in size than the lipid emulsion droplets (utilized for digestion, describedabove).The micelleshave a disk like shapewith lipids (monoacylglycerol,fatty acids, cholesteroland phospholipids)at the interiorand bile salts at the periphery.The hydrophilic groupsof the lipids are orientedto the outside (close to .the aqueous environment) and the hydrophobicgroupsto the inside.In thisfashion, the bile salt micellesexert a solubilizingeffect on the lioids. Stomach Almost unchanged Pancrealiclipase i PhospholipaseAz+L Cholesterylesterase L Fig. 8.11 : Overview of digestion of lipids.
  • T ; 176 BIOCHEMISTFIY INTESTINALMUCOSALCELL 7Phospholipid Fiq.8.12 : Absorptionof lipidsby intestinalmucosalcell. pqt*r.:haril}lsan*,S lipEr6absorption The mixed micelles serve as the major vehicles for the transport of lipids from the intestinal lumen to the membrahe of the intestinal mucosal cells, the site of lipid absorption.The lipid componentspassthrough the unstirred fluid layer and are absorbed through the plasma membrane by diffusion (Fig.8.l2). Absorption is almost complete for monoacylglycerolsand freefatty acidswhich are slightly water soluble. However, for water insoluble lipids, the absorption is incomplete. For instance, less than 4OY. of the dietary cholesterolis absorbed. The micelleformationis alsoessentialfor the absorptionof fat soluble vitamins, particularly vitaminsA and K. The efficiency of lipid absorption is dependent on the quantity of bile salts to solubilizedigestedlipids in the mixed micelles. It may, however, be noted that in the absenceof bile salts,the lipid absorptionoccursto a minor extent.This is mostly due to the slightlywater soluble nature of monoacylglycerolsand free fatty acids.Further,shortand medium chain fatty acidsarenot dependenton micelleformationfor the absorption. Synth:esEs of EiBids isl ttre [ratev*imal mucosa[ cells The fatty acids of short and medium chain length(< 10 carbons),aftertheir absorptioninto the intestinalcells, do not undergoany modi- fication. They enter the portal circulation and are transportedto the liver in a bound form to albumin. The long chain fatty acids are activated by thiokinase (fatty acyl CoA synthetase)in the intestinal cells. The acyl CoA derivativesso formed combine with 2-monoacylglycerolsto produce triacylglycerols. These reactions are catalysed by a group of enzymes, namely acyftransferases(Fig.8.13). Further, within the intestinal cells, cholesterol is converted to cholesterylester, and phospholipids are regenerated from the absorbed lysophospho- lipids. The newly synthesizedlipids are usually different from those consumed in the diet. $ecretion of lipids from the intestinal mucosal cells The lipids that are resynthesized(described above)in the intestinalcellsare hydrophobicin nature.They are put togetheras lipid droplets and surroundedby a thin layer consistingof mostly apolipoproteins (At and B-48) and phospholipids.This packageof lipidsenveloped in the layerstabilizesthe dropletsand increases their solubility. These particlesare known as chylomicrons. Chylomicrons migrate to the plasma membraneof intestinalmucosalcells.They are releasedinto the lymphaticvesselsby exocytosis.
  • Ghapter I : DIGESTIONAND ABSOFPTION 177 Fattyacid----Z--------+ Fattyaryl-CoA Acytfians{eras6 Acvttranslerase cholesterol-------------- -+ cholestervlester TV Fatty CoA acylCoA 2-Monoacylglycerol Cholesterol Shortchain fattyacids Portalcirculation Lymphaticsystem tBlood J Peripheraltissues Fig. 8,13 : Formation and secretion of chylomicronsby intestinal mucosalcells. The presenceof chylomicrons(Creek; chylos- juice) gives the lymph a milky appearance, which is observed after a lipid-rich meal. Chylomicronsenterthe largebody veinsvia the thoracic duct. Blood from hereflows to the heart and then to the peripheral tissues (muscle, adiposetissue)and, finally,to the liver.Adipose tissueand muscletake up a largeproportionof dietarylipidsfrom chylomicronsfor storageand transport. lt is believed that this bypass arrangement(passageof chylomicronsthrough peripheraltissues)protectsthe liver from a lipid overload after a meal. Abnorrnalities of lipid digestion and absorption The gastrointestinaltract possessesan efficient systemfor digestionand absorptionof lipids. lt can comfortablyhandleas much as 4 timesthe normaldaily intakeof lipids. Steatorrhea : lt is a condition characterized by the lossof lipids in the feces.Steatorrheamay be due to 1. A defect in the secretion of bile or pancreaticjuice into the intestine;
  • 178 BIOCHEMISTFIY 2. lmpairmentin the lipid absorptionby the intestinalcells. Steatorrheais commonly seen in disorders associatedwith pancreas,biliary obstruction, severeliver dysfunctionetc. #h*e *eii*,rr;i ! ai{q}n* "; Cholesterolstone formation in gall-bladder (gallstones)is a frequenthealthcomplication.lt isfoundmorefrequentlyin ferrralesthanin males often in associationwith obesity.Cholesterolgall stones are formed when liver secretes bile (containing phospholipids,bile acids etc.), supersaturatedwith respectto cholesterol. SBESITV AruffiFAT ABSORPTIOro Obesity is a major problem in many partsof the world as the availabilityof food is generally abundantand overeatingis common. Intakeof lipids largelycontributesto obesity.ln recent years,pharmacologicalinterventionsto prevent fat digestion,absorption,and thus obesityare in use.Two approachesare given below 1. Pancreatic lipase degrades dietary triacylglycerolto fatty acidsand glycerolwhich are absorbed. Orlistat is a non-hydrolysable analog of triacylglycerol,and is a powerful inhibitorof pancreaticlipase,hencepreventsfat digestion,and absorption. 2. Olestra is a syntheticlipid, produced by esterificationof naturalfatty acids with sucrose (insteadof glycerol).Olestratasteslike a natural lipid. However, it cannot be hydrolysedand therefore,getsexcreted. Nucleic acids (DNA and RNA), and their bases purines and pyrimidines can be synthesizedin the body, and thus they are dietarilynon-essential. BIOMEDTCAL/ CLINICAT CONCEPTS FS' $5 Cooking of food significantlyimprouesthe digestibilityby enzymes. Lactose intolerance due to a delect in the enzyme lactose(ftgalactosidase)is uery common. The treatment oduocatedis seuere restriction of lactose (milk and milk products)in the diet. Flatulence, occurring alter ingestion of certain non-digestible oligosaccharides,is characterizedby increosedintestinal motility, cramps and irritation. Direct intestinal absorptionof proteins and polypeptides is obseruedin the infants, immediatelyat'terbirth. This is importantfor the transferot'maternal immunoglobulins (uia breast-feeding)to the offspring. In some odults, macromolecular(protein) absorption by intestine is responsiblefor antiborlyformation, olten causingfood allergV. Emulsification ot' lipids is essenfiolfor their et't'ectiuedigestion,since iipcsescan act only on the surJaceof lipid droplets. Bile saltsare the mostet'ficientbiolqical emulsifyingagenia. Pharmacologicalinteruentions (e.g. Orlistat, Olestra) to block fat digestion ond/or absorptionso os fo preuent obesitgare in recent use. Steotorrhea,characterizedby the loss o/ lipids in t'ecesis commonlg ossociatedwith impaired pancreaticfunction and biliory obstruction. ag Gosfric ulcers are mainly coused by the bacterium H. pylori. The antibiotics that eliminate this bacterium are effectiuein the treatment. re Acute pancreatitis is cousedby autodigestionol pancreaswhile chronic pancreatiusis associatedwith excessiueconsumptionol alcohol.
  • Chapter a : DIGESTIONAND ABSOBPTTON Nucleicacids (DNA,RNA) Unchanged SmallIntestine Ribonu- cl€ases Deoxvribo- | nuclebses IPhosPhodiesterases + Nucleotides I pi*{ Nucleotidases + Nucleosides (Deoxv)riboseJ Nucleosidases + frurines.Pyrimrdrni,,s Stomach LowpH DNA,RNA denatured Fig. 8.14 : Overview of digestion of nucleic acids. The digestion of dietary nucleic acids is carried out in the small intestine,primarily by the enzymes of pancreatic juice. Ribonucleases and deoxyribonu cl eases,respectively, hydroIyse RNA and DNA to oligonucleotides(Fig.8.t4). The latteraredegradedby phosphodiesterasesto form mononucleotides.Nucleotidasesact on nucleotides to liberate phosphate and nucleosides.The nucleosides mav be either directly absorbed or degraded to free bases before absorption. Some of the unabsorbed purines are metabolized by the intestinal bacteria. The dietarypurinesand pyrimidinesare not of much utility for the synthesisof tissuenucleic acids.Further,the purinesaftertheir absorption aremostlyconvertedto uric acid by the intestinal mucosalcells and excretedin the urine. The following are the major abnormalities(of interestto biochemists)concernedwith digestion and absorptionof food in the gastrointestinal tract. Lactose intolerance,deficiency of sucrase, Hartnup'sdiseaseand steatorrheahave already beendescribed.Pepticulcerand pancreatitisare other important disorders associated with digestivesystem. Peptic ulcers Castric and duodenal ulcersare collectively known as peptic ulcers.Ulcerationoccurs due to the autodigestionof mucosa by the gastric secretions(pepsin and HCI). In the patients of peptic ulcer,gastricHCI is alwayspresentin the pyloric regions of stomach and the duodenum.Gastic ulcersare mainly causedby the bacterium Helicobacterpylori which lives in the nutrient-rich gastric mucosa, H. pylori induceschronic inflammationin the stomach tissues,which getsexposedto acid damage.For this reason, the best mode of treatment for gastric ulcers is the use of antihiotici that eliminate H. pylori. Achlorhydria is a less serious disorder involvingthe failure to secretegastricHCl. Pancreatitis Inflammationof the pancreasis known as pancreatitis.Acute pancreatitisis causedby the autodigestionof pancreasdue to the unusual conversionof zymogensinto the activeenzymes by trypsin. ln normal circumstances,this is preventedby trypsin inhibitor. Acute pancreatitis is a lifethreatening disorder.Measurementof serum amylase(highly efevated)is usedin the diagnosisof pancreatitis. Excessiveconsumption of alcohol over a long period is blamedas the prime causeof chronic pancreatitis.
  • 180 BIOCHEMISTFIY L Digestion is o process that conuertscomplex foodstufls into simpler ones which can be reodily absorbed b9 the gastrointestinal trsct. 2. Stomach,duodenum and upper part ol small intestine qre the maiot sitesof digestion. The small intestine is the prime site for the obsorption of digested foods. 3. The digestion of corbohydratesis initiated in the mouth by saliuary aamylase qnd is completed in the small intestine by pancreotic anmylase, oligosoccharidasesond disaccharidases. 4. Monosacchoridesare the final absorbable products of carbohydrate digestion. Glucose is tronsported into the intestinal mucosol cells by o carrier mediated, No+-dependent energy requiring process. 5. Lactoseintolerancedue to a defect in the enzyme loctose(ftgolactosidose)resultingln the inobitityto hydrolyselactow (mitk suEar)is the common abnormahtyof corbohydratedigestion' 6. Protein digestion begins in 1he stomach by pepsin, which is oided by gastric HCl. Pancreatic proteoses (trypsin, chymotrypsin and elostase) and intestinal amino' peptidasesand dipeptidasescomplete the degradation of proteins to amino ocidsand some dipeptides. 7. The intestinol absorption of amino ocids occurs by different transport systems(at least six known). The uptoke ol omino ocids is primarily a No+-dependentenergy requiring process. 8. Digestionof tipids occursin the small intestine. EmulslJicationol lipids, brought obout by bile solfs, is a prerequisitefor their digestion. Pancreatic lipose aided by a colipase degradestriacylglycerol to 2-monoacylglycerol andfree fatty acids.Cholesterol esterase and phospholipases,respectiuely, hydrolyse cholesteryl esters and phospholipids. 9. Lipid obsorption occursthrough mixed micelles,formed by bile sslfs in associstionwith pioducts ol lipid digestion (primarily 2-monoacylglycerol, cholesterol ond lree fatty acids). In the intestinol mucossl cetls, Iipids ore resynthesized Jrom the obsorbed components and packed as chylomicrons which enter the lymphatic uesselsand then the blood. 10. Dietary nucleic acids(DNA ond RNA) are digestedin the small intestine to nucleosides ond/or bases(purines and pyrimidines) which are absorbed.
  • Ghapter 8: DIGESTIONAND ABSOFPTION I. Essayquestions 1. Writean accountof the digestionand absorptionof lipids. 2. Describebrieflythe digestionof carbohydratesand proteins. 3. Cive an accountof the Na+dependentintestinaltranspoftof glucoseand aminoacids. 4. Describethe role of intestinein the digestionof foodstuffs. 5. Write briefly on the enzymesof gastrointestinaltract involvedin the digestionof foodstuffs. II. Short notes (a) Mixed micelles,(b) Lactoseintolerance,(c) Salivaryamylase,(d) Disaccharidases,(e) y- Clutamylcycle,(fl Zymogens,(g)Specificityof proteases,(h)Bilesalts,(i)Synthesisof chylomicrons in the intestinalmucosalcells,(i) Pancreaticjuice. III. Fill in the blanks 1. Celluloseisnotdigestedin humansdueto lackof theenzymethathydrolyses bonds. 2. Themostimportantcarbohydrateassociatedwith flatulencecausedby ingestionof leguminous seeds 181 3. 4. 5. 6. 7. Lactoseintoleranceis causedby the deficiencyof the enzyme The non-digestedcarbohydratesare collectivelycalled GastricHCI is secretedbv Nameof the peptidebelievedto be involvedin thetransportof aminoacids The disease characterized by impairment in the absorption of neutral amino acids 8. Trypsinhydrolysespeptidebonds,the carbonylgroupof which is contributedby the amino acids or 9. The inhibition of the enzyme pancreaticlipaseby bile salts is overcomeby a protein, namely 10. Thevehiclesfor the transportof lipidsfrom the intestinallumento the membraneof mucosal cells IV. Multiple choice questions 11. Transportof glucosefromthe lumento the intestinalmucosalcellsis coupledwith diffusionof (a)Na+(b) K+ (c)Cl- (d) HCOI. 12. The key enzymethat convertstrypsinogento trypsinis (a)Secretin(b) Chymotrypsin(c) Elastase(d) Enteropeptidase. 13. The productsobtainedby the actionof pancreaticlipaseon triacylglycerolsare (a)Clycerolandfreefattyacids(b)1-Acylglycerolandfreefattyacids(c)2-Acylglycerolandfree fatty acids(d) 3-Acylglyceroland free fatty acids. 14. The lipoproteinssynthesizedin the intestinalmucosalcellsfrom the absorbedlipidsare (a)Highdensitylipoproteins(b)Chylomicrons(c)Lowdensitylipoproteins(d)Verylow density lipoproteins. 15. Salivaryc-amylasebecomesinactivein the stomachprimarilydue to
  • PlasmaProteins Th" plasma is the liquid medium of blood | (55-60%),in which the cell components- namely erythrocytes, leukocytes, platelets-are suspended.lf blood containing anticoagulants (e.g.heparin,potassiumoxalate)is centrifuged, the plasmaseparatesout as a supernatantwhile the cells remainat the bottom.The packedcell volume or hematocritis about 45%. The term serum is applied to the liquid mediumwhich separatesout afterthe blood clots (coagulates).Serumdoes not contain fibrinogen and other clotting factors. Thus, the main difference between plasma and serum is the presence or absenceof fibrinogen. lFt's6l{,.ti,rrr*+ot $f ood The total volume of blood in an adult is around 4.5 to 5 liters. Blood performsseveral diversifiedfunctions.These include respiration, excretion, acid-base maintenance, water balance,transportof metabolites,hormonesand drugs,body defenseand coagulation. Separatlon eit plasma proteins The total concentrationof plasmaproteinsis about6-8 g/dl.The plasmais a complexmixture of proteins,and severaltechniquesareemployed to separatethem. An age-oldtechniqueis based on the use of varying concentrations of ammonium sulfateor sodium sulfate.By this method, which is known as saltingout process, the plasma proteins can be separated into three groups-namely albumin, globulins and fibrinogen. Electrophoresis: This is the most commonly employedanalyticaltechniquefor the separation of plasma(serum)proteins.The basicprinciples of electrophoresisare described in Chapter 43. Paper or agar gel electrophoresiswith vernol buffer (pH-8.6) separatesplasma proteins into 5 distinctbandsnamelyalhumin, a1, a2, B and y globulins (Fig.9.l). The concentration of each one of these fractionscan be estimatedby a densitometer. 182
  • Chapter 9: PLASMAPROTEINS 183 Fig.9.1 : Electrophoresisofplasmaproteins- ,i::,mliiftffi lffitl-ii{F ,tH 'j ' Abnormal electrophoretic pattern Electrophoresis of serum proteins is convenientlyused for the diagnosisof certain diseases 1. Multiple myeloma : A sharp and distinct M band appearsin the lglobulin fraction. 2. Acute infections : c1- and a2- globulins are increased. 3. Nephroticsyndrome: Decreasedalbumin with sharpand prominentc,2-globulin. 4. Primary immune deficiency : Diminished yglobulin band. 5. a1-Antitrypsindeficiency: Diminishedcr1- globulinband. Albumin/globulin (A/G) ratio : The albumin concentrationof plasmais 3.5 to 5.0 g/dl while that of total globulins is 2.5 to 3.5 g/dl. The normal A/G ratio is 1.2 to 1.5 : 1. The A,/Cratio is loweredeitherdue to decreasein albumin or increasein globulins,as found in the following conditions 1. Decreasedsynthesisof albumin by liver- usuallyfound in liverdiseasesand severeprotein malnutrition. 2. Excretionof albumin into urine in kidney damage. 3. Increased production of globulins associated with chronic infections, multiple myelomasetc. Gomponents of plasma proteins The important plasma proteins along with their characteristics(based on electrophoretic pattern) and major functions are given in Table 9.1. Some selected plasma proteins are discussedhereunder. Albumin is the major constituent(600/o)ol plasmaproteinswith a concentrationof 3.5-5.0 g/dl. Human albumin hasa molecularweight of 69,000, and consistsof a single polypeptide chain of 585 amino acids with 17 disulfide bonds. Synthesis of albumin Albumin is exclusivelysynthesizedby the liver. For this reason, measurementof serum albumin concentrationis convenientlyused to assessIiverfunction (synthesisdecreasedin liver diseases).Liverproducesabout 12 g albuminper day which represents25'/. of the total hepatic proteinsynthesis.Albumin hasan half-lifeof 20 days. Functions of albumin Plasmaalbumin performsosmotic, transport and nutritivefunctions 1. Osmotic function : Due to its high concentration and low molecular weight, albumin contributes to 75-8oo/oof the total plasma osmotic pressure(25 mm Hg). Thus, albuminplaysa predominantrole in maintaining blood volume and body fluid distribution. Decreasein plasma albumin level resultsin a fall in osmotic pressure,leading to enhanced fluid retentionin tissuespaces/causingedema. The edema observed in l<washiorkor,a disorder Stan Globulins
  • 184 BIOCHEMISTF|Y Plasma Molecular concentration weight Maior function(s) Albumin Prealbumin 3.5-5.0g/dl 2$-30mg/dl 69,000 61,000 Osmotic,transport,nutritiveandbuffering Transportsthyroxinetosomeextent a,-Globulins c,-Antitrypsin c[1-Lipoproteins(HDL) Orosomucoid Retinolbindingprotein(RBP) Thyroxinebindingglobulin(TBG) 54,000 Inhibitoroftrypsin Transportscholesterolandphospholipids 44,000 Bindswithprogesterone 21,000 TransportsvitaminA 58,000 Transportsthyroidhormones 52,000 Majortransporterofsteroidhormones(e.9. cortisol,corticosterone) 0.3.{,5g/dl < 0.2g/dl 0.2{.3 g/dl < 0.1g/dl 3-6 mg/dl 1-2 mg/dl 3-4 mg/dlTranscortinorcortisol bindingprotein(CBG) oq-Globulins o9-Macroglobulin Haptoglobins (Hp1-1;Hp2-1andHp2-21 Prothrombin Ceruloplasmin 0.4-0.8g/dl 0.2{.3g/dl < 0.3g/dl < 0.02g/dl < 0.03g/dl 800,000 Antitrypsinandantiplasminactivity 90,000 Bindswithplasmafreehemoglobinand preventsitsexcretion 63,000 Participatesinbloodcoagulation 150,000 Transportofcopper;oxidationofFe2*toFe$. p-Globullns p-Lipoproteins(LDL) Transfenin Hemopexin Plasminogen 0.6-1.1g/dl 0.2{.5g/dl 0.24.3g/dl < 0.1g/dl <0.05g/dl Transportstriacylglycerolsandcholesterol 76,000 Transportsiron 57,000 Transportsheme 140,000 Formsplasmin,involvedinfibrinolysis yGlobullns 0.8-1.8mg/dl Antibodyfunctions (fmmunoglobulins-lgG,lgA,lgM,lgDandlgE;relerTable9.2lordetails) 0.2-0.4g/dl 340,000 ParticipatesinbloodcoagulationFibdnogen of protein-energymalnutrition, is attributed to a drasticreductionin plasmaalbumin level. 2. Transport functions : Plasma albumin binds to several biochemically important compounds and transports them in the circulation. These include free fatty acids, bilirubin,steroidhormones,calciumand copper. [Note : Besidesalbumin, there are several other plasma transport proteins. These include prealbumin, retinol binding protein, thyroxine binding protein,transcortinand othersas stated in the functionsof plasmaproteinsin Table9.11. 3. Nutritive functions : Albumin servesas a sourceof amino acidsfor tissueproteinsynthesis to a limited extent, particularly in nutritional deprivationof amino acids. 4. Buffering function : Among the plasma proteins,albumin has the maximum buffering
  • f ira!"r!;*s"S : PLASMA PFIOTEINS 18s capacity. However, the buffering action of albuminin plasmais not significantcomparedto bicarbonatebuffersystem. ,'t ;I::;,-:,! $Ea;,{,fiIf *{:en{:t'* *,}{ o*!*eet:'i:i;,,;; 1. Albumin,bindingto certaincompoundsin the plasma, preventsthem from crossingthe blood-brain barrier e.B. albumin-bilirubin complex, albumin-freefatty acid complex. 2. Hypoalbuminemia (lowered plasma albumin)is observedin malnutrition,nephrotic svndromeand cirrhosisof liver. 3. Albumin is excreted into urine (albuminuria) in nephrotic syndrome and in certaininflammatoryconditionsof urinarytract. Microalbuminuria (3O-3O0mg/day) is clinically importantfor predictingthe future risk of renal diseases(Refer Chapter 36). 4. Albumin is therapeuticallyusefulfor the treatmentof burns and hemorrhage. Globulinsconstituteseveralproteinsthat are separatedinto four distinctbands(cr1,42, p and 1-globulins)on electrophoresis(See Fig.9.l). Clobulins, in general, are bigger in size than albumin. They perform a variety of functionswhich includetransportand immunity. ln Table9.1, the importantglobulinsare given, some of them are discussedhereunder. r t .3'1}5{Y,r u1-Antitrypsin,more recentlycalledas a-anti- proteinase,isa glycoproteinwith 394 aminoacids a,rda molecularweight of 54,000. lt is a major constituentof u1-globulin fraction of plasma :"oteinswith a normalconcentrationof about200 -q, dl. crl-Antitrypsinisa serineproteaseinhibitor. : combines with trypsin, elastaseand other :"o:easeenzymesand inhibitstheir activity. ' - :,iri:11.-.::;:-n{:t+ ' i" vt*5U*t,'* a. -{ntitrypsindeficiencyhasbeenimplicated - :,ro diseases,namely, emphysemaand a1-AT &ficiengy liver disease. Emphysema(Greek: emphusan-to inflate)is a term usedto representthe abnormaldistension of lungsby air. At least5% of emphysemacases are due to the deficiency of a1-AT. This is associatedwith lung infections(e.g.pneumonia) and increasein the activity of macrophagesto releaseelastasethat damageslung tissues.In the normal circumstances, elastase activity is inhibitedby a1-AT. Effectof smokingon crl-AT : The amino acid methionineat position358 of a1-ATis involved in binding with proteases.Smoking causes oxidation of this methionine to methionine sulfoxide.As a result, a1-AT with methionine sulfoxide cannot bind and inactivate proteases. Emphysemais more commonly associatedwith heavysmokingand the situationbecomesworse in personswith cr1-AT deficiency. cl1-Antitrypsindeficiency and liver disease: This is due to the accumulationof a mutant o1-AT which aggregatesto form polymers.These polymers,in turn-by an unknown mechanism- cause liver damage (hepatitis) followed by accumulationof collagen resultingin fibrosis (cirrhosis). ix]-11#flc FsffiBrok$Eiigr It is a high molecular weight (8,00,000) proteinand is a major constituentof u,2-fraction. cr2-Macroglobulininhibits proteaseactivity and servesas an anticoagulant.lts concentrationin plasma is elevated in nephrotic syndrome. This is due to the fact that majority of the low molecular weight proteins are lost in urine (proteinuria)in this disorder. HAPTOGLOBIN Haptoglobin(Hp) is a plasmaglycoprotein with an approximate molecular weight of 90,000. Hp is an acute phaseprotein since its plasma concentration is increased in several inflammatoryconditions. *;c,!yle;;td'i1'rt$ a;S!'*,4p'i r;e6 d# A;rige Haptoglobinbinds with the free hemoglobin (known as extra-corpuscularhemoglobin)that spills into the plasma due to hemolysis.The
  • 186 d BIOCHEMISTRY haptoglobin-hemoglobin (Hp-Hb) complex (mol. wt. 155,000)cannot passthrough glomeruli of kidney while free Hb (mol. wt. 65,000) can. Haptoglobin, therefore,preventsthe lossof free Hb into urine. Clinical significance of Hp : Hemolytic anemia is associatedwith decreasedplasma concentrationof haptoglobin.This is explained as follows. The haltt-life of Hp is about 5 days while that of Hp-Hb complex is 90 min. ln hemolyticanemia,free Hb in plasmais elevated leading to increased formation of Hp-Hb complex. This complex, in turn, is rapidly clearedfrom the plasmaresultingin decreased Hp levels. CERULOPLASMIN Ceruloplasminis a blue colou;'ed,copper- containinga2-globulinwith a molecularweight of 150,000.lts plasmaconcentrationis about 30 mg/dl. Ceruloplasminbindswith almost9O"/"oI plasma copper (6 atoms of Cu bind to a molecule).This bindingis rathertight and, as a result,copper from ceruloplasminis not readily releasedto the tissues.Albumin carrying only 1)oh of plasmacopper is the major supplierof copper to the tissues.Ceruloplasminpossesses oxidase activity, and it is associated with Wilson's disease which is discussed under copper metabolism (Chapter 1A. TRANSFERRIN Transferrin (T0 is a glycoprotein with a molecularweight of 76,000.lt is associatedwith p-globulinfraction.Tf is a transporterof iron in the circulation. ACUTE PI{ASE PROTEINS Acute phaseresponserefersto a non-specific responseto the stimulus of infection, injury, variousinflammatoryconditions(affectingtissue/ organs),canceretc.Thisphaseis associatedwith a characteristicpattern of changes in certain plasmaproteins,collectivelyreferredto as acute phaseproteinse.g.o,1-antitrypsin,ceruloplasmin, complementproteins,C-reactiveprotein.During the acute phase, synthesisof certain plasma 45678910fi12 Days ----* Fig.9.2 : The responseof C-reactiveprotein (CRP)in response to surgery (The normal acute Phase is depicted proteins decreases,and they are regardedas negative acute phase reactantse.g. albumin, transferrin. C-reactive Frotein (CRPI CRP is a maior component of acute phase proteins.lt is producedin the liverand is present in the circulation in minute concentration (< 1 mgidl). C-reactiveprotein (C strandsfor carbohydrateto which it bindson the capsuleof pneumococi) is involved in the promotion of immune system through the activation of complementcascade. Estimationof CRP in serum is importantfor the evaluation of acute phase response.The response of CRP to surgery is depicted in Fig.9.2. ln a normal surgery, serum CRP increasesand returns to normal level within 7-10 days.lf the recoveryis complicatedby any infection,it will be reflectedby the continuous elevation of CRP which requires further treatment. The higher vertebrates,including man, have evolved a defensesystemto protect themselves against the invasion of foreign substances-a virus, a bacterium or a protein. The defense o) E IE o E a by blue line, the development ot infection by red line and the responseafter treatment by green line).
  • Chapter 9 : PLASMA PFIOTEINS 187 *HrN lnterchain disulfidebonds S-S S_ rNHs *iirN 1- NH-r Fab Hingeregion cHo Intrachain disulfidebonds cH2 Fc t-"'= F-', coo- strategiesof the body are collectivelyreferredto as immunity, and are briefly described under immunology(Chapter42). lmmunoglobulins(or antibodies)are describedhere. lmrmunoglobulins-basic concepts lmmunoglobulins,a specialisedgroup of proteins are mostly associated with y-globulin fraction(on electrophoresis)of plasmaproteins. Someimmunoglobulinshowever,separatealong with P and a-globulins.Therefore,it should be noted that y-globulin and immunoglobulin are not synonymous. lmmunoglobulin is a functional ferm while y-globulin is a physical term. Structure of isnmunoglobulins All the immunog:lobulin (Ig) molecules oasigally consist of two identical heavy (H) chains(mol.wt. 53,000to 75,000each)andtwo identicallight (L chains(mol. wt. 23,000 each) held togetherby disulfide linkagesand non- covalent interactions(Fi9,9.3.Thus, immuno- globulin is a Y-shapedtetramer (HzLz). Each heavy chain containsapproximately450 amino acids while each light chain has 212 amino acids. The heavy chains of Ig are linked to carbohvdrates,hence immunoglobulins are glycoproteins. Constant and variable regions : Each chain (L or H) of lg hastwo regions(domains),namely the constant and the variable. The amino terminal half of the light chain is the variable region(V1)while the carboxyterminalhalf is the constant region (Cg).As regardsheavy chain, approximatelyone-quarterof the amino terminal regionis variable(Vx) while the remainingthree- quartersis constant(Cs,, Csr, Csr). The amino
  • 188 BIOCHEMISTFIY Type H-Chain L{hains Molecular Percentage werght carbohydrate Itaior fundion(s)Molecular formula Serumconc, mC/dl IgG rorl, y2r2or y2)r2 -150,000 80f1,500 Mostlyresponsiblefor humoralimmunity IgA rorl, (o2rj1aor -(160,000h-i (oraldr+ 15G.400 Protectsthebody surfaces IgM rorl, - 900,000 12 50-200 Humoralimmunity, servesasfirstlineof defense p2rj5 or ItzT"zls IgD rorl, (Qr2orQt2) -180,000 13 1-10 B-cellreceptor? IgE rorl, E2K2Ol e2?u2 -190,000 12 0.02-0.05 Humoralsensitivity andhistaminerelease, acid sequence (with its tertiary structure)of variable regionsof light and heavy chains is responsible for the specific binding of immunoglobulin(antibody)with antigen. Proteolytic cleavage of Ig : An immuno- globulin can be split by the enzyme papain to their fragments.Theseare two identicalantigen binding fragments(Fab)and one crystallizable fragment(Fc).Papaincleavesthe immunoglobin molecule at the site between Cp,1 and Cp2 regionswhich is referredto as hinge region. CLASSESOF IMMUNOGLOBULINS Humans have five classes of immuno- globufins-namely IgG, IgA, IgM, IgD and IgE-containing the heavychainsy, c, p, 6 and E, respectively. The type of heavy chain ultimatelydeterminesthe classand the function of a given lg. Two typesof light chains-namely kappa (r) and lambda(l.)-are found in immunoglobulins. They differ in their structurein C1 regions.An immunoglobulin(of any class)containstwo K or two l, light chains and never a mixture. The occurrenceof r chains is more common in human immunoglobulinsthan l, chains. The characteristicsof the 5 classesof human immunoglobulinsare given in Table 9.2. lmmunoglobulin G (IgGl IgC is the most abundant(75-80%) classof immunoglobulins.IgC is composedof a single Y-shapedunit (monomer).lt can traverseblood vesselsreadily.IgG is the only immunoglobulin that can cross the placenta and transfer the mother'simmunity to the developingfetus.IgG triggers foreign cell destruction mediated by complementsystem. Innmunoglobulin A {fgA} IgA occursas a single(monomer)or double unit (dimer)held togetherby J chain. lt is mostly found in the body secretionssuchassaliva,tears, sweat,milk and the walls of intestine.IgA is the mostpredominantantibodyin the colostrum,the initial secretionfrom the mother's breastafter a baby is born. The IgA molecules bind with bacterial antigenspresenton the body (outer epithelial) surfacesand remove them. In this way, IgA prcvents the foreign substancesfrom enteringthe body cells. lrnmunoglobulin M (IgM! lgM is the largestimmunoglobulincomposed of 5 Y-shapedunits (IgC type) held togetherby a J polypeptidechain. Thus IgM is a pentamer. Due to its large size, IgM cannot traverseblood vessels,hence it is restrictedto the blood stream.
  • Ghapter 9 : PLASMAPROTEINS 189 IgM is the first antibody to be produced in responseto an antigen and is the most effective against invading microorganisms. lt may be notedthatIgM can simultaneouslycombinewith 5 antigenicsitesdue to its pentamericstructure. Immunoglobulin D (IgD! IgD is composedof a single Y-shapedunit and is present in a low concentrationin the circulation.IgD moleculesare presenton the surfaceof B cells.Theirfunction,however,is not known for certain. Some workers believe that IgD may function as B-cell receptor. lmmunoglobulin E (IgEl IgE isa singleY-shapedmonomer.lt isnormally presentin minuteconcentrationin blood.IgE levels are elevatedin individualswith allergiesas it is associatedwith the body's allergicresponses.The IgE moleculestightlybind with mastcellswhich releasehistamineand causeallergy, Production of ;mmunoglobulins by multiple Eenes As alreadydiscussed,immunoglobulinsare composedof light and heavy chains.Eachlight chain is producedby 3 separategenes,namely a variable region (V1)gene/ a constantregion (Cl) gene and a joining region (J)gene. Each heavy chain is produced by at least4 different genes-a variable region (Vg) gene, a constant region (Cg) gene, a joining region U) gene and diversityregion (D) gene. Thus multiple genes are responsiblefor the synthesisof any one of the immunoglobulins. Antibody diversity : A person is capable of generatingantibodies to almost an unlimited range of antigens(more than one billion!). lt should, however, be rememberedthat humans do not contain millions of genesto separately code for individualimmunoglobulinmolecules. The antibodydiversityis achievedby two special processes/ namely comhination of various structural genes and somatic mutations, MULTIPLE MYELOMA Multiple myeloma, a plasma cell cancer, constitutesabout1'/" of all cancersaffectingthe population. Femalesare more susceptiblethan malesfor this disorderand it usuallyoccurs in the age group 45-60 years. Abnormal lg production : Multiple myelomais dueto the malignancyof a singlecloneof plasma cells in the bone marrow. This resultsin the overproductionof abnormal immunoglobulins, mostly (75%) IgG and in some cases(25"h)IgA or IgM. IgD type multiple myeloma found in youngeradults is lesscommon (<2%) but more severe. In patients of multiple myeloma, the synthesis of normal immunoglobulins is diminishedcausingdepressedimmunity. Hence recurrentinfectionsarecommonin thesepatients. Electrophoretic pattern : The plasma of multiplemyelomapatientsshowsa characteristic patternof electrophoresis.There is a sharpand distinct band (M band, for myeloma globulin) betweenp-andy-globulins.Further,this M band almost replacesthe y-globulinband due to the diminishedsynthesisof normal y-globulins. Bencefones proteins : Henry BenceJonesfirst described them in 1847. These are the light chains (r or l,) of immunoglobulinsthat are synthesizedin excess.BenceJonesproteinshave a molecular weight of 20,000 or 40,000 (for dimer).In about 2O"/oof the patientsof multiple myeloma, Bence .fonesproteins are excreted in urine which often damagesthe renal tubules. Amyloidosis is characterizedby the deposits of light chain fragments in the tissue (liver, kidney, intestine)of multiple myeloma patients. The presenceof BenceJonesproteinsin urine can be detected by specific tests. 1. Electrophoresis of a concentratedurine is the best test to detect Bence Jones proteins in uflne. 2. The classical heat test involves the precipitation of Bence Jones proteins when slightlyacidifiedurine is heatedto 40-50'C. This precipitateredissolveson further heatingof urine to boiling point. lt reappearsagain on cooling urine to about 70oC. 3. Bradshaw'stest involves layering of urine on concentratedHCI that forms a white ring of precipitate, if Bence Jonesproteins are present.
  • 190 ., BIOCHEMISTRY lntrinsic patnway I{z Extrinsic parhway I FactorX Prothrombint,lftThro bin(ra) Fibrinogen(l) Fibrin (bloodclot) Fig.9.4: Overuiewof blood cloningwith the finalcommonpathway- The term hemostasis is applied to the sequence of physiological responsesto stop bleeding(lossof blood after an injury).This is carriedout by blood clotting. Blood clotting or coagulationis the body's major defense mechanism against blood loss.A blood clot is formed as a resultof a seriesof reactions involving nearly 20 different substances,most of them being glycoproteins, synthesized by the liver. Blood clotting process involves two independentpathways 1. The extrinsicpathwayisthe initialprocess in clotting and involvesthe factorsthat are not presentin the blood (hencethe name). 2. The intrinsic pathway involves a seriesof reactions participated by the factors present in the blood. Strictly speaking,the extrinsic and intrinsic pathwaysare not independent,since they are coupledtogether.Further,the final reactionsare identical for both pathwaysthat ultimately lead to the activationof prothrombinto thrombinand the conversion of fibrinogen to fibrin clot lFig.e.4). The blood coagulation factors in human plasma along with their common names and molecularweightsare listedin Table9"3.All but two of thesefactorsare designatedby a Roman numeral.lt should, however,be noted that the numbersrepresentthe order of their discovery and not the orderof their action.The cascadeof blood clotting processis depicted in Fig.9.5 and the salient featuresare discussedbelow. The active form of a factor is designatedby a subscript a. The active clotting factors (with exceptionof fibrin) are serineproteases. Sonversion of librinogen to fibrin Fibrinogen(factorl) is a solubleglycoprotein that constitutes2-3"/"of plasmaproteins(plasma concentration0.3 g/dl). Fibrinogenconsistsof 6 polypeptide chains-two A cr,two B p and two 1 making the structure(A ct)z(B F)z'lz. Fibrinogen undergoes proteolytic cleavage catalysed by thrombin to release small fibrinopeptides (A and B), This results in the formation of fibrin monomerswhich can stick together to form hard clots (Fig.9.6). Clot formationis furtherstabilizedby covalentcross- linking betweenglutamineand lysineresidues. This reaction cross-linksfibrin clots and is catalysedby fibrin stabilizingfactor (Xlll).The red colour of the clot is due to the presenceof red cells entangledin the fibrin cross-links. Conversion of prothromhin to thronnbin Prothrombin(ll) is the inactivezymogenform of thrombin (lla).The activationof prothrombin
  • Chapter I : PLASMAPROTEINS 191 occurson the plateletsand requiresthe presence of factorsVa and Xa, besidesphospholipidsand Ca2+. The extrinsic pathwas* The extrinsic pathway is very rapid and occurs in responseto fissueinjury. This pathway essentiallv involves the conversion of proconvertin(Vll)to itsactiveform (Vlla)and the generation factor Xa. The tissue factor (lll), found to be necessaryto acceleratethe action Vlla on a factorX, is presentin lung and brain. The intrinsic pathway The intrinsic pathway is rather slow. lt involvesthe participationof a contact system (wounded surface) and a series of factors to generatefactor Xa. The Hagemanfactor(Xll)is activated(Xlla)on exposureto activatingwound surfacecontaining collagen or plateletmembranes.The formation of Xlla is acceleratedby kallikrein and HMK. The activated Hageman factor (Xlla) activates factor Xl. The Xla activate the Christmasfactor (lX). The Christmasfactor is also activatedby active proconvertin(Vlla). In the next step, the Staurt factor (X) is activated by Christmas factor (lXa) and this reactionrequiresthe presenceof antihemophilic factor (Vllla), Ca2+and phospholipids. The extrinsicand intrinsic pathwayslead to the formation of factor Xa which then participatesin the final common pathway to ultimatelyresultin the formationof fibrin clot. Anticoagulants Severalsubstances,known as anticoagulants, are in useto inhibitthe blood clotting.Calcium is essentiallyrequired for certain reactionsof blood coagulation.The substanceswhich bind with Ca2+are very effectiveas anticoagulants. These include oxalate, fluoride, EDTA and citrate. Heparin is an anticoagulantusedto maintain normal hemostasis.lt is a heteropolysaccharide found in manytissuesincludingmastcellsin the endotheliumof blood vessels.Heparincombines with antithrombinlll which in turn. inhibitsthe Factor number Common name(s) Subunit molecular weight I tl ill IV V vtl vill IX X XI xtl xill Fibrinogen Prothrombin Tissuefactor,thromboplastin Calcium(Ca2*) Proaccelerin,labilelactor Proconvedin,serumprothrombinconversionaccelerator(SPCA) AntihemophilicfactorA,antihemophilicglobulin(AHG) Christmasfactor,antihemophilicfactorB, Plasmathromboplastincomponent(PTC) Staurt-Prowerfactor Plasmathromboplastinantecedent(PTA) Hagemanfactor Fibrin-stabilizingfactor(FSF),fibrinoligase,LikiLorandfactor Prekallikrein Highmolecularweightkininogen(HMK) 340,000 720,000 370,000 330,000 50,000 330,000 56,000 56,000 160,000 80,000 320,000 88,000 150,000 Note: ThenunbersrepresenttheoderoftheirdisnveryaN nottheorderofthehaction.FactorVawason@refenedtoasfactorVl, hencethereisnQfactorVl.
  • 192 BIOCHEMISTFIY FactorXll Prekallikrein FactorXl FactorlX FactorVl||----;*-=-+ VlI lrilr'li:!.)n |J;Ll.liilrJV Ertrinsic i pathway i Vlla+-,------: FactorVlIi Factorlll FactorX Thrombin(lla) Fibrinogen(l) ractoJxttt---.------+ xtt Fibrin (hardclot) Fig.9,5 : Theblood clottingcascadein humans (the active forms of the factors are reprcsented in red with subscript'a'). clotting factorsll, lX, X, Xl, Xll and kallikrein. Heparin can be administeredto patientsduring and after surgeryto retard blood clotting. The blood containsanother anticoagulant- namely protein C-which is activated by thrombin. Active protein C hydrolyses and inactivatesclotting factorsV and Vlll. Warfarin, a vitamin K antagonist may be consideredasan oral anticoagulanf.This actsby reducingthe synthesisof certainclottingfactors (ll, Vll, lX and X). i";nlEr"ls*olneii*, The term fibrinolysisrefersto the dissolution or lysis of blood clots. Plasmin is mostly responsiblefor the dissolution of fibrin clots. Plasminogen,synthesizedin the kidney, is the inactive precursor of plasmin. Tissue plasminogen activator (TPA) and urokinase convert plasminogento plasmin. Streptokinase is a agentwhichactivates therapeutic fibrinolytic plasminogen. FlMnogen II ThrombinI l9 Fibrinope AandB ItrY _./------_/-------_-/ Fibrinmonorner Y Flbrinclot Flg.9.6: Diagrammaticrepresentationof fibrinclotformationfromfibrinogen.
  • frrr:r:,tslr ii : PLASMA PBOTEINS 193 i:.l4f1+."+:riii l+.l rt', i i.rir:::i'1: :',r.:i':' Severalabnormalitiesassociatedwith blood clotting are known. Theseare due to defectsin clotting factors which may be inherited or acquired.Hemophilia,Von Willebrand'sdisease etc., are examplesof inherited disorder while afibrinogenemiais an acquireddisease. HemophiliaA (classicalhemophilia): This is a sex-linked disorder transmitted bv females affecting males. Hemophilia A is the most common clotting abnormalityand is due to the deficiency of antihemophilic factor (VIil). The affectedindividualshaveprolongedclottingtime and sufferfrom internalbleeding(particularlyin joints and gastrointestinaltract).HemophiliaA has gained importancedue to the fact that the Royalfamiliesof Britainare amongthe affected individuals. Hemophilia B (Christmasdisease): This is due to the deficiency of Christmasfactor (lX). The clinicalsymptomsare almostsimilarto that found in hemophiliaA. Von Willebrand's disease: This disorder is characterizedby failureof plateletsto aggregate and is due to a defect in the olateletadherence factor. BIoMEDTCAL/ GUNTCALCONCEPTS rx' Albumin, the most abundant plasma protein, is inuolued in osmotic t'unction, transport of several compounds (fqtty actds, steroid hormones), besidesthe bulfering action. !ii Hypoalbuminemia qnd albuminuria are obseruedin nephrotic syndrome. uyAntitrypsin deliciency has been tmplicated in emphysema (abnormal distension ol lungs by qir) which is more commonly associotedwith heauy smoking. Haptoglobin preuentsthe possiblelossof free hemoglobin trom the plasma through the kidneys by lorming hoptoglobin-hemoglobin complex. Immunoglobulins (antibodies), a specialized group of plasma globulor proteins, are actiuely inuolued in immunity. lgG and lgM are primarily concerned with humoral immunity while IgE is ossociofed with allergic reactions. Multiple myeloma, a plasmacell cancer diseoseof bone marrow, is characterlzedbg ouerproduction ol abnormal immunoglobulins (mostly lgG). Laborotory diognosis ol multtple myelomo can be made by the presenceof a distinct M band on plasmo/serum electrophoresis, Blood clotting or coagulation is the body's major d.efense mechanism against blood loss. Delects in clotting factors cause coagulation abnormalities such [.t" as hemophllia A (det'lciency of lactor VIII) qnd Christmos diseose (deficiency of foctor IX). st Anticosgulants Inhibit blood clotttng. These include heparin, qxalate, fluoride, EDTA and citrate. Ii1
  • BIOCHEMISTRY 194 .a '1F.ftt1iI";F..q' L The total concentrationof plosmaproteinsis about 6-8 g/dl. Electrophoresisseporotes plasma proteins into 5 distinct bqnds, namely albumin, a1, &2, p and y globulins' 2'Albuministhemajorconstituent(60%)oJplosmoproteinswithaconcentrationS'5to 5.0g/d|.Itisexclusiuelysynthesizedbgtheliuer,Albumtnperformsosmotic,transport ond nutritiue functiorts. 3. ayAntitrgrpsinis a major constituent ot'.al globul^,!liil',"i^^?:-Antitrvpsin deficiencv his been implicated in emphysemaand a speciJicliuer dtsease' 4'Haptoglobin(Hp)bindswithfreehemoglobin(Hb)thatspillsintotheplasmadueto hemolysis.TheHp-Hb complex connot passthrough the glomeruli,hencehaptoglobin preuentsthe lossof t'reehemoglobininto urtne' 5. Alterationsin the acute phase proteins(e.g' ayantitrypsin, ceruloplasrnin'C'reoctiue protein)are obseruedos o result oJ non-spicit'ii responseto the stimulus of infection' injury,inflommationetc'EstimationofserumC-reactiueproteinisusedforthe euqluotionot' acutephaseresponse' 6. Immunoglobulins ore specializedproteins to defend the body ogoinst the forergn subsfonces.They are mostlgassociatedwith yglobulin t'ractionof plasmaproteins' The. immunoglofullinsessenfiollgconsist ol two ilentical heauy choinsand two identical Iight chains,held together by disult'ide linkages' 7. Fiuecrossesof immunogroburins-namery IgG, IgA, lgM., IgD.and lgE-<re t'ound-in humans.IgG is most aiundant and is ^ioi"tv reiponsiblet'or .humoral immunitg. lgA protectsbodg surt'aces.lgM seruesos a t'irsttineoi d"1"nt" t'or humoral immunitg while IgE is associated with allergic reactions' S.Multip|emgelomaisduetothemalignancyot'asingleclo-ne^ot'plasmacellsinthebone marroLu.This couses the ouerproduction oJ abnormal lgG- .The plosma of multiple myeloma patients on electrophore-sisshous a distinct M-band' 9. Blood clottingis the bodg's maiordefense mechanismagainstblood loss' The extrinsic and intrinsic pathwaysleadto the formation of factorxa which then participotes in .the final common pathway to actiuate prothrombin to thrornbin' Fibrinogen is then conuertedto librin clot. lO. Plasmin is mosfly responsibleJor the dissolufion of fibrtn synthesizedby the kidieg, is the inactiue precursor of plasmin' clots. Plasminogen, Tissueplosminogen ictiuator (TPA) and urokinase conuert plosminogen to plasmin'
  • Ghapter9 : PLASMAPFIOTEINS t95 [. Essayquestions 1. Describethe characteristicsand majorfunctionsof plasmaproteins. 2. Give an accountof differenttypesof immunoglobulinsalongwith theirfunctions. 3. Discussthe cascadeof blood clottingprocess. 4. Describethe structureof differentimmunogloublins. 5. Discussthe roleof acutephaseproteinsin healthand disease. II. Slrort notes (a) Electrophoresisof plasmaproteins,(b)Functionsof albumin,(c)at-Antitrypsin,(d)Haptoglobin, (e) lmmunoglobulinC, (0 Multiple myeloma, (g) Bencejones proteins, (h) Fibrinogen, (i) Anticoagulants,(j) Hemophilia. III. Fill in the blanks 1. The differencebetweenplasmaand serumis the presenceor absenceof 2. The mostcommonlyemployedtechniquefor separationof piasmaproteins 3. Haptoglobinbindsand preventsthe excretionof the compound 4. The cellsresponsiblefor the productionof immunoglobulins 5. The immunoglobulinthat can crossthe placentaand transferthe mother'simmunityto the developingfetus 6. The immunoglobulinsthatcan bind with mastcellsand releasehistamine 7. Bence-Jonesproteinsare precipitatedwhen urine is heatedto 8. The majorcomponentof acutephaseproteinsusedfor the evaluationof acutephaseresponse 9. The extrinsic and intrinsic pathwaysresult in the formation of a common activated factor 10. The factormostlyresponsiblefor the lysisof blood clot IV. Multiple choice questions 11. HemophiliaA is due to the deficiencyof clottingfactor (a)X (b) V (c)VIll (d) ll. 12. Plasmaalbuminperformsthe followingfunctions (a)Osmotic(b)Transport(c) Nutritive(d)All of them' 13. The immunoglobulinpresentin mostabundantquantity (a)lgc (b) lsA (c) IBM(d) leE. 14. Namethe immunoglobulininvolvedin body allergicreactions (a)lgA (b) lgE(c) lgD (d) lgM. 15. The followinganticoagulantbindswith Ca2+and preventsblood clotting (a)Heparin(b)oxalate (c) Proteinc (d)All of them.
  • HemoglobinandPorphyrins The hemoglobin sgeahs : "I arn rheredof blood,respowibleJbr respirntion; DeliuerA, to tissuesand return CO, n lungs; InfluencedbyfactorspII, BPGand.Cl- in rnyfunctiens; Disttn'bedin mydutiesbystru.entralahnormalities," Th" structure,functionsand abnormalitiesof I hemoglobin,the synthesisand degradation of heme, the porphyrin containingcompounds are discussedin this chapter. Hemoglobin (Hb) is the red blood pigment, exclusively found in erythrocytes (Creek; erythrose-red; kytos-a hollow vessel).The normal concentrationof Hb in blood in males is 14-16 B/dl, and in females 13-15 B/dl. Hemoglobin performstwo importantbiological functionsconcernedwith respiration 1. Delivery of 02 from the lungs to the tissues. 2. Transportof CO2 and protonsfrom tissues to lungsfor excretion. Hemoglobin(mol.wt. 64,450)is a conjugated protein, containing globin-the apoprotein Flg. 10.1: Diagrammaticrepresentationof hemoglobin with2u and2fl chains(Redblocks-Heme). part-and the heme-the non-protein part (prostheticgroup). Hemoglobin is a tetrameric allosteric protein (Fig.I0.l). Structureof globin : Clobin consistsof four polypeptide chains of two different primary structures(monomericunits).The common form of adult hemoglobin(HbAl) is made up of two a-chainsand two p-chains(o.2P).Someauthors considerhemoglobinconsistingof two identical dimers-(ap)1 and (cr0)2.Eacha-chain contains 14'l amino acids while p-chain contains 146 amino acids. Thus HbA, has a total of 574 196
  • Ghapter 1O: HEMOGLOBINAND POFIPHYFIINS 197 FIg. 102 : 9tructureof hene IM-MeW f CH); V-VrnVl ',,,'.(rlS'flalrii,'o,#fi ff.,t'ffi9-,flF-,ff,fi r-.S',.f'a-,..--'..9',fffifidr't. amino acid residues. The four subunits of hemoglobinare held togetherby non-covalent interactionsprimarily hydrophobic,ionic and hydrogenbonds.Eachsubunitcontainsa heme 8roup. Structure of heme : The characteristic red colour of hemoglobin(ultimatelyblood) is due to heme. Heme containsa porphyrin molecule namely protoporphyrin lX, with iron at its center.ProtoporphyrinlX consistsof four pyrrole rings to which four methyl, two propionyl and two vinyl groupsare attached(Fig.l0.A. Heme is common prostheticgroup presentin cytochromes, in certain enzymes such as catalase,tryptophan pyrolase,and chlorophyll (Mg2*). In case of cytochromes,oxidation and reduction of iron (fe2* rr Fe3+)is essentialfor their biological function in electron transport chain. Other forms of hemoglobin Besides the adult hemoglobin (HbAl) describedabove, other minor hemoglobinsare also found in humans(Tahle l0.l). ln adults a small fraction (< 5%) of hemoglobin,known as HbA2 is present. HbA2 is composed of two a and two 6 (defta)chains. Fetalhemoglobin (HbF) is synthesizedduring the fetal developmentand a little of it may be present even in adults. Glycosylated hemoglobin (HbA1), formed by covalentbindingof glucoseis alsofound in low concentration.lt is increasedin diabetesmellitus which is successfullyutilizedfor the prognosisof these patients (details under Diabetes, in Chapter 36). Myoglobin Myoglobin (Mb) is monomeric oxygen binding hemoproteinfound in heartand skeletal muscle.lt has a singlepolypeptide(153 amino acids)chainwith hememoiety.Myoglobin(mol. wt. 17,000)structurallyresemblesthe individual subunits of hemoglobin molecule. For this reason, the more complex properties of hemoglobinhave been convenientlyelucidated throughthe studyof myoglobin. Myoglobin functions as a reservoir for oxygen. lt further seryesas oxygen carrier that promotesthe transportof oxygento the rapidly respiringmusclecells. Functions of hemoglobin Hemoglobin is largely responsiblefor the transportof 02 from lungsto tissues.lt alsohelps to transportCO2 from the tissuesto the lungs. Binding of O" to hemoglobin One molecule of hemoglobin (with four hemes)can bind with four moleculesof 02. This is in contrast to myoglobin (with one heme) which can bind with only one molecule of oxygen. In other words, each heme moiety can bind with one 02. TyPe Composition and Percentageof symbol total hemoglobin HbAl HbA2 HbF HbAlc az1z %62 sz^[z o2Fr-glucose 90% < 3-/o < 2o/o < 5'/o l-i: )11;'f'-V ,N-PM Histidineot (/ ) globin ll HN*cH,
  • f- 198 BIOCHEMISTFIY 50 pO2(mmHg) Fig. 10.3 : Orygen dissociation cuNes of hemoglobin and myoglobin (pOr- Partial prcssure of orygen). Oxygen dissociation curve : fhe hinding ability of hemoglohin with 02 at differentpartial pressuresof oxygen (pO2) can be measuredby a graphicrepresentationknown as 02 dissociation curve.The curvesobtainedfor hemoglobinand myogfobinare depicted in Fig.l0.3. It is evident from the graph that myoglobin has much higher affinity for 02 than hemoglobin. Hence 02 is bound more tightly with myoglobinthan with hemoglobin.Further, pO2 neededfor half saturation(50% binding)of myoglobinis about'l mm Hg comparedto about 26 mm Hg for hemoglobin. Cooporative binding $f 0s to hemrogiobin Theoxygendissociationcurvefor hemoglobin is sigmoidal in shape (Fig.l0.3).This indicates thatthe bindingof oxygento one hemeincreases the binding of oxygento other hemes.Thus the affinityof Hb for the last02 is about 100 times greaterthan the binding of the first 02 to Hb. This phenomenon is referred to as cooperative binding of 02 to Hb or simply heme-heme interaction(Fig.l0.4. On the otherhand,release of 02 from one heme facilitatesthe releaseof 02 from others. ln short, there is a communication among heme groups in the hemoglobinfunction. Transport of O" to the tissues In the lungs,wherethe concentrationof 02 is high (hencehigh pO2),the hemoglobingetsfully saturated(loaded)with 02. Conversely,at the tissuelevel,where the 02 concentrationis low (hence low pO2), the oxyhemoglobinreleases (unloads)its 02 for cellular respiration.This is often mediated by binding 02 to myoglobin which servesas the immediate reservoirand supplierof 02 to the tissues(Fig.l0.5). T and R forms of hemoglobin The four subunits(clzpz)of hemoglobinare held together by weak forces. The relative position of these subunits is different in oxyhemoglobincomparedto deoxyhemoglobin. T-form of Hb : The deoxy form of hemoglobin existsin a T or taut (tense)form. The hydrogen and ionic bonds limit the movement of monomers.Therefore,the T-form of Hb has low oxygen affinity. R-formof Hb : The bindingof 02 destabilizes some of the hydrogen and ionic bonds particularlybetweenaB dimers.This resultsin a relaxed form or R-form of Hb wherein the AI o -c = Eso 6 (' U) s 100 Q2 Increaslngaflinityfor 02
  • Chapter 1O: HEMOGLOBINAND PORPHYRINS 199 subunitsmove a little freely. Therefore, the R-form has high oxygen affinity. The existenceof hemoglobin in two forms (T and R) suitably explains the allosteric behaviour of hemoglobin (Fig.t0.a). Transport of CO2 by hemoglobin ln aerobic metabolism, for every moleculeof 02 utilized,one moleculeof CO2 is liberated. Hemoglobin actively participatesin the transportof CO2 from the tissuesto the lungs.About 15'/" of CO2 carriedin blood directlybindswith Hb. The rest of the tissue COz is transportedas bicarbonate(HCO3). Carbondioxide moleculesare bound to the uncharged cr-amino acids of hemoglobin to form carbamyl hemoglobinas shown below Hb- NH2+ CO2$ Hb- NH-COO-+ H+ TheoxyHb can bind 0.15 molesCO2l mole heme,whereasdeoxyHb can bind 0.40 molesCO2lmoleheme.Thebinding of CO2 stabilizesthe T (taut) form of hemoglobin structure, resulting in decreased02 affinityfor Hb. Hemoglobin also helps in the transport of CO2 as bicarbonate, as explainedbelow (Fig.l0.6). As the CO2 entersthe blood from tissues,the enzyme carhonic anhydrase present in erythrocytescatalysesthe formationof carbonic acid (H2CO3).Bicarbonate(HCOJ) and proton (H+) are releasedon dissociationof carbonic acid. Hemoglobin acts as a buffer and immediatelybinds with protons.lt is estimated that for every 2 protons bound to Hb, 4 oxygen molecules are releasedto the tissues.In the lungs,binding02 to Hb resultsin the releaseof protons.The bicarbonateand protonscombine to form carbonic acid. The latter is acted upon by carbonicanhydraseto releaseCO2,which is exhaled. BOHR EFFECT The binding of oxygen to hemoglobin decreaseswith increasing H+ concentration (lower pH) or when the hemoglobinis exposed to increasedpartialpressureof CO2 (pCOz).This phenomenonis known as Bohreffect.lt is due to a change in the binding affinity of oxygen to hemoglobin. Bohr effect causes a shift in the oxygen dissociation curve to the right (Fig.t0.V. Bohr effect is primarily responsiblefor the releaseof 02 from the oxyhemoglobinto the tissue.This is becauseof increasedpCO2 and decreasedpH in the activelymetabolizingcells. NHCOO- CarbamylHb Oxy lr" / / NHCOO- O2 2 Fe Fe Fe ,- TrssuEs Myoglobin1 Fig. 10.5 : Diagrammatic representation of
  • 200 BIOCHEMISTFIY 2CO2+ 2H2O T caroonic J anhVdrase 2H2COs Exhaled tI 2CO2+2H2Q t 2H2CO3 1 2H++ 2HCO! -_-YJ LUNGS plasma. In order to maintain neutrality, Cl- enters the erythrocytes and binds with deoxyhemoglobin.The concen- tration of Cl- is greater in venous blood than in arterial blood. The four substances namely2,3-bisphosphoglycerate (describedbelow),CO2, H+ and Cl- are collectivelycalled as allosteric effectors. Thev interact with the hemoglobin molecule and facilitate the release of 02 from oxy- hemoglobin. * 40z Mechanism of Bohr effect The Bohr effectmay be simplifiedas follows HbO2 + H+ -+ Hb H+ + 02 Any increasein protons and/or lower pO2 shifts the equilibrium to the right to produce deoxyhemoglobinas happensin the tissues.On the other hand, any increasein pO2 and / or a decreasein H+ shiftsthe equilibriumto the left, which occursin lungs. When CO2 binds to hemoglobin,carbamyl hemoglobinis produced(detailsdescribedunder transportof CO2). This causesthe removal of protons from the terminal NH2 group and stabilizesthe structureof Hb in the T form (deoxyhemoglobin).Therefore,the binding of CO2 promotesthe releaseof oxygen(in tissues). On the other hand, when hemoglobin is oxygenatedin lungs,CO2 is releasedas it binds looselywith R-formof Hb. Role of Cl- in oxygen transport Chloride(Cl-) is bound moretightlyto deoxy- hemoglobin than to oxyhemoglobin. This facilitatesthe releaseof 02 which is explained as follows Bicarbonate (HCO3) is freely permeable across the erythrocyte membrane. Once produced in the erythrocytes, HCOJ freely movesout and equilibrateswith the surrounding EFFECT OF 2,3.B|SPHOSPHO. GLYCERATE ON (,2 AFFINITY OF Hb 2,3-Bisphosphoglycerate(2,3-BPG;formerly, 2,3-diphosphoglycerate)is the most abundant organicphosphatein theerythrocytes.ltsmolar concentrationisapproximatelyequivalentto that of hemoglobin.2,3-BPC is produced in the erythrocytesfrom an intermediate(1,3- bisphosphoglycerate)of glycolysis.This short pathway, referredto as Rapaport-Leubering cycle,is describedin carbohydratemetabolism (Chapterl3). 50 pO2(mmHg) N o = o E50 (u @ s Fig. 10.7 : Effect of pH (Bohr effect) on oxygen dissociation curue (pOr-Partial pressure ot Or).
  • GhapterlO : HEMOGLOBINAND PORPHYFIINS 201 StrippedHb (no2, 3-BPG) 100 N o = o E50 6 a s 0 50 pO2(mmHg) Flg. 10.8: EftectotpH (Bahrettect)onorygen .,,,,,,tF,F,#tF'I!L9,{F-,,,QF,'I:F:;PiPffP,f:,Pd,t::,,,,,,:';, Binding of 2,3-BPG to deoxyhemoglobin 2,3-BPC regulates the binding of 02 to hemoglobin.lt specificallybindsto deoxyhemo- globin (and not to oxyhemoglobin) and decreasesthe 02 affinity to Hb. The effect of 2,3-BPCon Hb mav be summarizedas follows HbO2 + 2,3-BPC ------sHb-2,3-BPC + 02 oxYHb "","JtTJr:J"o The reducedaffinity of 02 to Hb facilitates the releaseof 02 at the partialpressurefound in the tissues.This 2,3-BPC shifts the oxygen dissociationcurve to the right (Fi9.10.0. Mechanism of action of 2'3-BPG One molecule ol 2,3-BPC binds with one molecule (tetramer)of deoxyhemoglobinin the centralcavity of the four subunits.This central pocket has positively charged (e.g. histidine, lysine)two p-globin chains. lonic bonds (salt bridges) are formed between the positively charged amino acids (of p globins) with the negativelychargedphosphategroupsof 2,3-BPC (Fig.l0.9. The bindingof 2,3-BPCstabilizesthe deoxygenated hemoglobin (T-form) by cross- linkingthe p-chains. {-Blood of anemicoatient (2,3-BPGT) On oxygenationof hemoglobin,2,3-BPC is expelledfrom the pocketand the oxyhemoglobin attainsthe R-formof structure. Glinical significance of 2,3-BPG Since the binding of 2,3-BPC with hemoglobin is primarily associatedwith the releaseof 02 to the tissues,this small molecule assumesa lot of biomedical significance.The erythrocytelevelsof 2,3-BPCare relatedto tissue demandsof oxygen supply. 1. ln hypoxia: The concentrationof 2,3-BPC in erythrocytesis elevated in chronic hypoxic conditions associated with difficulty in O2 supply. These include adaptation to high altitude, obstructive pulmonary emphysema (airflow in the bronchiolesblocked)etc. 2. ln anemia : 2,3-BPC levels are increasedin severeanemia in order to cope up with the oxygendemandsof the body.This is an adaptationto supply as much 02 as possibleto the tissue,despitethe low hemoglobinlevels. 3. In blood transfusion: Storageof blood in acid citrate-dextrosemedium results in the decreasedconcentrationof 2,3-BPC.Suchblood when transfusedfailsto supply02 to the tissues immediately. Addition ol inosine(hypoxanthine-ribose)to the stored blood preventsthe decreaseof 2,3- BPC. The ribose moiety of inosine gets phosphorylated and enters the hexose monophosphate pathway and finally gets convertedto 2.3-BPC. Normalblood (with2,3-BPG) 100 O.t ,0 -c. o ttl H-C-O-P-O- tl H-C-H O- I oI O=P-O- I o- (B)(A) Fig. 10.9: (A) Diagrammatic reprcsentation of binding of 2,3-BPG to deoxyhemoglobin; (B) Structure of 2,3-BPG.
  • BIOCHEMISTRY 202 4. Fetal hemoglobin(HbF) : The binding of 2,3-BPC to fetal hemoglobin is very weak' Therefore, HbF has higher affinity for 02 comparedto adult hemoglobin(HbA).This may be needed for the transfer of oxygen from the maternalblood to the fetus. Hemoglobin (specificallyheme) combines with different ligands and forms hemoglobin derivatives.The normal blood containsoxyHb and deoxyHb. Besides these, methemoglohin (metHb) and carboxyhemoglobin are the other important Hb derivatives'The Hb derivatives have characteristiccolour and they can be detected by absorPtionspectra. Methernoglobin Forthe biologicalfunctionof hemoglobin-to carry oxySen-the iron should remain in the ferrous(Fe2+)state.Hemoglobin (Fe2+)can be oxidized to methemoglobin(Fe3+).In normal circumstances,however,molecularoxygendoes not oxidize Hb, it only loosely binds to form oxyhemoglobin. The oxidation of hemoglobin to methemoglobin(metHb)may be causedin the living system by H2O2, free radicals and drugs' The methemoglobin(with Fe3+)is unableto bind to 02. lnstead,a water molecule occupiesthe oxygen site in the heme of metHb. tn normal circumstances,the occasional oxidation of hemoglobin is corrected by the enzyme methemoglobin reductase present in erythrocytes(Fig.l0.1A. Carboxyhemogiobin {COHbl Carbon monoxide(CO) is a toxic compound (an industrialpollutant)that can bind with Hb in the samemanneras02 binds.However,CO has about 200 times more affinity than 02 for bindingwith Hb. Clinical manifestationsof CO toxicity are observedwhen the COHb concentrationexceeds 20"/".fhe symptoms include headache,nausea, Ftg, 10.10: Conuercionof hemoglobinto methemoglobin and vice versa' breathlessness,vomitingand irritability'Adminis- trationof 02 throughoxygenmaskswill help to reversethe manifestationsof CO toxicity' Abnormal hemoglobinsare the resultantof mutations in the genes that code for a or p chains of globin' As many as 400 mutant hemoglobinsare known.About 95% of them are due to alterationin a singleamino acid of globin' Basic concepts of globin synthesis For a better understanding of abnormal hemoglobins,it is worthwhile to have a basic knowledgeof globin synthesis'The globin genes are organisedinto two genefamiliesor clusters (Fig.l0.tl). 1. o-Gene family : There are two Senes coding for a-globin chain presenton each one of chromosome 'l6. The (-gene, other member of a-gene clusteris also found on chromosome 16 and is active during the embryonic development. 2. p-Genefamily : The synthesisof p-globin occursfrom a singlegene locatedon each one of chromosome11. This chromosome also contains four other genes. One e-geneexpressedin the early stagesof embryonicdeveloPment. Hemoglobin (Fe2*) NADH+ H+
  • Ghapter 1O: HEMOGLOBINAND PORPHYHINS 203 azlz o262 aZFZ rttttl 768 o-Globinlikegenes (chromosome16) Globinchains Hemoglobins Globinchains p-Globin-likegenes (chromosome11) Gy Fig. 10.11: Diagrammaticrepresentationof globin genes with the synthesisof globin chains and hemoglobins(("er-Hb Gower 1;a;yr-HbF; arSSHbA; arBr-HbA,). Two ygenes (Gy and Ay) synthesizey-globin chainsof fetal hemoglobin(HbF). One 6-geneproducing6-globin chain found in adultsto a minor extent (HbA2). l{emoglobinopathies It is a term used to describethe disorders causedby the synthesisof abnormalhemoglobin molecule or the production of insufficient quantitiesof normal hemoglobinor rarelyboth. Sickle-cell anemia (HbS) and hemoglobin C disease(HbC) are the classical examples of abnormal hemoglobins. Thalassemias,on the otherhand,arecausedby decreasedsynthesisof normalhemoglobin. Sickle-cellanemia(HbS)is the mostcommon form of abnormal hemoglobins.lt is so named becausethe erythrocytesof these patientsadopt a sickle shape (crescentlike) at low oxygen concentration (Fig.l0.1A. Occurrence of the disease Sickle-cellanemia is largely confined to tropicalareasof the world. lt primarilyoccursin the black population. lt is estimatedthat 1 in 500 newborn black infants in the USA are affectedby sickle-cellanemia. Molecular basis of HbS The structureof hemoglobin (as described already)containstwo cr-andtwo p-globinchains. In case of sickle-cellanemia, the hemoglobin (HbS)hastwo normal a-globin chainsand two abnormal (mutant)p-globin chains.This is due to a differencein a singleamino acid. In HbS, glutamate at sixth position of p-chain is replacedby valine (Clu pu-+ Val). Sickle-cell anemia is due to a change (missensemutation) in the single nucleotide (thymine-+ adenine)of p-globingene.Thiserror causesthe formationof alteredcodon (CUC in Fig. 10.12: Erythrocytes: (A)Froma normalperson; (B)Froma patientol sickelcellanemia. (B)(A)
  • 204 BIOCHEMISTFIY -cTc- t_GAG_ J*HN-CH-CO,,% I CHz t- QHz I coo- (-Gtu*) HemoglobinA -cAc- _GUG_ J *HN-CH-CO ,,t.",..,...* Aminoacid I CH / HsC CHs (- !/6[ * ) F-Chain6thposirion HemoglobinS DNA RNA(codon) FIg. 10,13 : Formation of B-chain of hemoglobin in normal and sickle cell anemia (Note : Single base mutation in DNA(T -+ A ) causesreplacementof glutamateby valineat 6th positionof B-chain). placeof CAG) which leadsto the incorporation of valine instead of glutamate at the sixth position in p-chain (Fig.l0,l3). Homozygousand heterozygousHbS : Sickle- cell anemiais saidto be homozygous,if caused by inheritanceof two mutant genes(one from each parent)that code for p-chains.In case of heterozygousHbS, only one gene (of p-chain) is affected while the other is normal. The erythrocytesof heterozygotescontain both HbS and HbA and the diseaseis referredto as sickle- cell trait which is more common in blacks (almost 1 in 10 are affected).The individuals of sickle-cell trait lead a normal life, and do not usually show clinical symptoms. This is in contrast to homozygous sickle-cell anemta. Abnormalities associated with HbS Sickle-cell anemia is characterizedbv the following abnormalities 1. Life-longhemolytic anemia : The sickled erythrocytesare fragife and their continuous breakdownleadsto life-longanemia. 2. Tissuedamageand pain : The sickledcells block the capillariesresultingin poor blood supplyto tissues.This leadsto extensivedamage and inflammationof certaintissuescausingpain. 3. Increased susceptibility to infection : Hemolysisand tissuedamageare accompanied by increased susceptibility to infection and diseases. 4. Prematuredeath: Homozygousindividuals of sickle-cell anemia die before they reach adulthood(< 20 years). Mechanism of sickling in sickle-cell anemia Clutamateis a polar amino acid and it is replacedby a non-polarvaline in sickle-cell hemoglobin.This causesa marked decreasein the solubilityof HbS in deoxygenatedform (T- form). However,solubilityof oxygenatedHbS is unaffected. Sticky patches and formation of deoxyhemoglobin fibres The substitutionof valinefor glutamateresults in a stickypatchon the outersurfaceof p-chains. It is presenton oxy- and deoxyhemoglobinS but absent on HbA. There is a site or receptor complementaryto sticky patch on deoxyHbS. The stickypatchof one deoxyHbSbindswith the receptor of another deoxyHbS and this processcontinuousresultingin the formationof long aggregate molecules of deoxyHbS (Fi9.10.1a1.Thus, the polymerizationof deoxy- HbS moleculesleadsto long fibrousprecipitates (Fig.l0.15). These stiff fibres distort the erythrocytesinto a sickle or crescent shape (Fig.l0.lA. The sickled erythrocytesare highly vulnerableto lysis. ln case of oxyHbS, the complementary receptoris masked,althoughthe stickypatch is
  • Ghapten1O : HEMOGLOBINAND POFIPHYFINS 205 Fig. 10.14: Diagrammaticrepresentationofstickypatch(Blue)andstic$ patchreceptor( >) in thefomationoflongaggregatesof deoxyhemaglobins. present (Fi9.1O.14).Hence, the molecules of oxyHbS cannot bind among themselvesor with the moleculesof deoxyHbS. Normal deoxyHbA lacks sticky patchesbut contains receptors.Absence of sticky patches does not allow the deoxyHbA to participatein the formationof aggregates. As explained above, sickling is due to polymerizationof deoxyHbS.Therefore,if HbS is maintainedin the oxygenatedform (or with minimumdeoxyHbS),sicklingcan be prevented. Sickle-cell trait prov:des resistance to malaria The incidenceof sickle-celldiseasecoincides with the high incidenceof malariain tropical areasof the world (particularlyamongthe black Africans). Sickle-celltrait (heterozygousstatewith about 40% HbS)providesresistanceto malariawhich is a major causeof death in tropical areas.This is explainedas follows 1. Malaria is a parasiticdiseasecausedby Plasmodium falciparum in Africa. The malarial parasite spends a part of its life cycle in erythrocytes.lncreased lysis of sickled cells ishorterlife span of erythrocytes)interruptsthe oarasitecvcle. 2. More recentstudiesindicatethat malarial parasiteincreasesthe acidityof erythrocytes(pH down by 0.4). The lowered pH increasesthe sickling of erythrocytesto about 40% from the normally occurring 2Y". Therefore,the entry of malarial parasitepromotessickling leadingto lysis of erythrocytes. Furthermore, the concentrationof K+ is low in sickledcellswhich is unfavourablefor the parasiteto survive. Sickle-celltrait appearsto be an adaptation for the survivalof the individualsin malaria- infested regions. Unfortunately, homozygous individuals,the patientsof sickle-cellanemia (much lessfrequentthan the trait),cannot live beyond 20 years. Fig. 10.15: Diagrammaticrepresentation a fibreof aggregateddeoxyhemoglobin. DeoxyHbS
  • 206 BIOCHEMISTF|Y Biagnosis of sickfe.cell anemia 1. Sicklingtest : This is a simplemicroscopic examinationof blood smearpreparedby adding reducing agents such as sodium dithionite. Sickled erythrocytescan be detected under the mrcroscope. 2. Electrophoresis : When subjected to electrophoresisin alkaline medium (pH 8.6), sickle-cell hemoglobin (HbS) moves slowly towards anode (positive electrode) than doesadult hemoglobin(HbA).The slow mobility of HbS is due to less negativecharge,caused by the absence of glutamate residues that carry negative charge. In case of sickle-cell trait, the fast moving HbA and slow moving HbS are observed. The electrophoresisof hemoglobin obtained from lysed erythrocytes can be routinelyusedfor the diagnosisof sickle- cell anemiaand sickle-celltrait (Fig.t0.16). ifianaEenrent of sickle.cell disease Administration ol sodium cyanate inhibits sickling of erythrocyteg Cyanate increasesthe affinityof 02 to HbSand lowersthe formationof deoxyHbS. However, it causes certain side- effectslike peripheralnervedamage. In patients with severe anemia, repeated blood transfusionis required.This may resultin iron overloadand cirrhosisof liver. Replacementof HbS with other forms of hemoglobinshas been tried. Fetal hemoglobin (HbF)reducessickling.Sickle-celldiseaseawaits gene-replacementtherapy! Hemoglobin G disease Cooley'shemoglobinemia(HbC) is characte- rized by substitutionof glutamateby lysine in the sixthpositionof p-chain.Due to the presence of lysine, HbC moves more slowly on electrophoresiscomparedto HbA and HbS,HbC diseaseoccursonly in blacks.Both homozygous and heterozygousindividualsof HbC diseaseare known. This diseaseis characterizedby mild hemolytic anemia. No specific therapy is recommended. Normal Sickle-cellSickle-cell trait anemia mn II -..Origin.-'.-"-'--' @ Fig. 10.16 : Electrophoresis of hemoglobins at pH 8.6 (HbA-Normal adult hemoglobin; HbS-Sickle cell hemoglobin). Hemoglobin D This is causedby the substitutionof glutamine in place of glutamatein the 121stpositioinof B-chain.Severalvariantsof HbD are identified from different places indicated by the suffix. For instance,HbD (Punjab),HbD (LosAngeles). HbD, on electrophoresismoves along with HbS. Hemoglobin E This is the most common abnormal hemoglobinafterHbS. lt is estimatedthat about 1O% of the population in South-EastAsia (Bangladesh, Thailand, Myanmar) suffer from HbE disease.In India, it is prevalentin West Bengal. HbE is characterized by replacement of glutamate by lysine at 26th position of p-chain. The individualsof HbE (either homozygousor heterozygous)have no clinical manifestations. Thalassemiasare a group of hereditary hemolyticdisorderscharacterizedby impairment/ imbalancein the synthesisof globin chainsof Hb. Thalassemias(Greek: thalassa-sea)mostly occur in the regions surrounding the Mediterranean sea, hence the name. These diseases,however,are also prevalentin Central Africa, India and the Far East.
  • Chapter 1O: HEMOGLOBINAND POBPHYRINS 207 Molecular basis of thalassemias The basicconceptsin the synthesisof globin chains have been described (See Fig.l0,l41. Hemoglobincontains2a and 2p globin chains. The synthesis of individual chains is so coordinated that each a-chain has a p-chain partner and they combine to finally give hemoglobin (o"z!). Thalassemias are characterized by a defect in the production of a-or B-globin chain. There is however, no abnormalityin the amino acidsof the individual chains. Thalassemiasoccur due to a varietv of moleculardefects 1. Cene deletionor substitution, 2. Underproductionor instabilityof mRNA, 3. Defectin the initiationof chain synthesis, 4. Prematurechain termination. u-Thalassemias o,-Thalassemiasare caused by a decreased synthesis or total absence of a-globin chain of Hb. Thereare four copiesof a-globin Bene,two on each one of the chromosome16. Fourtypes of cr-thalassemiasoccur which deoend on the numberof missinga-globin genes.The salient featuresof differenta-thalassemiasare given in Table 10.2. 1. Silent carrier stateis due to lossof one of the four a-globin genes with no physical manifestations. 2. a-Thalassemiafraif causedby lossof two genes(bothfrom the samegenepairor one from each genepair).Minor anemiais observed. 3. HemoglobinH disease,due to missingof threegenes,is associatedwith moderateanemia. 4. Hydrops fetalis is the most severeform of a-thalassemiasdue to lack of all the four genes. The fetus usually survivesuntil birth and then dies. B.Thalassemias Decreased synthesis or total lack of the formation of p-glohin chain causes P- thalassemias.The productionof u-globin chain continuesto be normal,leadingto the formation of a globin tetramer(o4) that precipitate.This causes premature death of erythrocytes.There are mainly two types of p-thalassemias (Fig.t0.17) Typeof thalassemia Number of Schematicrepresentation missinggenes of geneson chromosome16 Clinical symptoms Normal Silentcanier a-Thalassemiatrait (heterozygousform) HemoglobinHdisease Hydropsfetalis flfl- -gt-El- --::giiG fl-g- -.-*i..r-El--,.01.,€F -'.-9i.€l-- ---';.-ql.i-EF -..9i.,r-&F --%-, f- Nil Nosymptoms Minoranemia Mildtomoderateanemia mayleadnormallife. Fetaldeathusually occursatbirlh. Nil J
  • 208 BIOCHEMISTF|Y Genesfor p-globinchain -r----t------- _/ 1pChain pThalassemia minor i:....r:..:::i No-pchain pThalassemia malor Fig. 10.17:Diagrammatictepresentationof gene deletionsin p-thalassemias (eachone of the chromosomepair of 11 hasone gene forB-globin). 1. p-Thalassemiaminor : This is an hetero- zygousstatewith a defect in only one of the two p-globin gene pairs on chromosome 11. This disorder, also known as p-thalassemiatrait, is usuallyasymptomatic,sincethe individualscan make some amount of p-globin from the affectedgene. 2. p-Thalassemia major : This is a homozygousstatewith a defectin both the genes responsiblefor B-globin synthesis.The infants born with p-thalassemiamajor are healthy at birthsincep-globinis not synthesizedduringthe fetal development. They become severely anemicand die within 1-2 years.Frequentblood transfusionis requiredfor thesechildren.This is associatedwith iron overloadwhich in turn may lead to death within 15-20 years. Porphyrinsare cyclic compoundscomposed of 4 pyrrole rings held together by methenyl (:CH-) bridges(Fig.l0JA. Metal ionscan bind with nitrogen atoms of pyrrole rings to form complexes. Heme is an iron-containing porphyrin (SeeFi9.10.2)while chlorophyll is a magnesium-containingporphyrin. Thus heme and chlorophyll are the classicalexamplesof metalloporphyrins. Fresentation and nomenclature of porphyrins Naturally occurring porphyrins contain substituentgroups replacing the 8 hydrogen atomsof the porphyrin nucleus. Hans Fischer, the father of porphyrin chemistry, proposed a shorthand model for presentationof porphyrinstructures.Accordingly, each pyrrole ring is representedas a bracket. Thus porphyrin has 4 closed bracketswith the I substituentoositionsnumberedas shown in Fig.l0.t8. Type I porphyrins : When the substituent groups on the 8 positionsare symmetrically arrangedthey are known as type I porphyrins, e.g. uroporphyrinL Typelll porphyrins: Theycontainasymmetric Broupsat the 8 positionsand are more common in the biological system.Originally, Fischer placed them as lX serieshence they are more popularlyknown astype lX porphyrins.lt may be observedthat the structureof uroporphyrin is asymmetric since on ring lV, the order of substituentgroups is reversed(P, A insteadof A, P). The Fischer'sshorthandmodelsof important porphyrins(uroporphyrinI and lll; coproporphyrin I and lll; protoporphyrinlX and heme) are depictedin Fig.l0.l9. Forphyrins in cancer therapy The photodynamicpropertiesof porphyrins can be used in the treatmentof certaincancers. This is carriedout by a techniquecalled cancer phototherapy. Tumors are capable of taking up The structureof porphyrins hasfour pyrrole rings namelyl, (c2oH14N4) lll and lV.
  • Chapter 1O: HEMOGLOBINAND POFIPHYFIINS 209 HC_CH ltll Hc"'r-cH H HH Poiphyrln 12 Fischer's model Fig. 10.18: Structuresofpynoleandporphyrin fllV arepynolerings;1-Bare substituentpositions; a, F,y,6arqn?tlly"lqne(-Cll=) bridgos,l rrore porphyrinsthan normaltissues.The cancer phototherapyis carried out by administering hematoporphyrin (or other related compounds) to the canceroatient.When the tumor isexoosed to an argonlaser,the porphyrinsget excitedand oroducecvtotoxiceffectson tumor cells. 65 Porpt4pin AP PA Uroporphyrln I AP PA Uroporphyrin lll MP PM Coproporphyrin I MP PM Coproporphyrin lll MV PM Protoporphyrln lX (lll) MV PM Heme FIg.10,19:Fischer'sshorthandmodelsat
  • 210 BIOGHEMISTFIY Heme is the most important porphyrin containingcompound.lt is primarilysynthesized in the liver and the erythrocyte-producingcells of bone marrow(erythroidcells).Hemesynthesis also occurs to some extent in other tissues. However, mature erythrocytes lacking mitochondria are a notable exception. Biosynthesisof heme occurs in the following stages(Fig.l0.20). 1. Formation of 8-aminolevulinate: Glycine, a non-essentialamino acid and succinyl CoA, an intermediatein the citric acid cvcle, are the starting materials for porphyrin synthesis. Glycine combines with succinyl CoA to form 6-aminolevulinate(ALA).This reactioncatalysed by a pyridoxal phosphate dependent 6-amino- levulinate synthaseoccurs in the mitochondria. It is a rate-controllingstepin porphyrinsynthesis. 2. Synthesisof porphobilinogen : Two mole- cules of 6-aminolevulinatecondense to form porphobilinogen(PBG) in the cytosol. This reactionis catalysedby a Zn-containingenzyme ALA dehydrafase.lt is sensitiveto inhibition by heavv metalssuch as lead. 3. Formation of porphyrin ring : Por- phyrin synthesisoccurs by condensation of four moleculesof porphobilinogen.The four pyrrole rings in porphyrin are interconnected by methylene (-CH2) bridges derived from cx,-carbonof glycine. The interaction of two enzymes-namely uroporphyrinogenI synthaseand uroporphy- rinogen lll cosynthase-resultsin condensation of porphobilinogenfollowed by ring closureand isomerizationto produce uroporphyrinogenlll. 4. Conversion of uroporphyrinogen lll to protoporphyrinlX : This is catalysedby a series of reactions (a) Uroporphyrinogendecarboxylasedecarbo- xylatesall the four acetate(A) sidechains to form methyl groups (M), to produce coproporphyrinogen. (b)Coproporphyrinogen oxidase convefts (oxidative decarboxvlation)two of the propionatesidechains(P)to vinyl groups (V) and resultsin the formationof proto- porphyrinogen. (c) Protoporphyrinogen oxidase oxidizes methylene groups (-CH2-) interconnectingpyrrolerings to methenyl groups (=CH-). This leads to the synthesisof protoporphyrinlX. 5. Synthesisof heme from protoporphyrin lX : The incorporati,onof ferrousiron (Fe2+)into protoporphyrinIX is catalysedby the enzyme ferrochelatase or heme synthetase.This enzyme can be inhibited by lead. lt is found that the induction of Fe2+ into protoporphyrin lX can occur spontaneouslybut at a slow rate. Regulation of heme synthesis Heme production in the liver is requiredfor the formationof hemoproteins(e.9.cytochrome P456involved in detoxification)while in the erythroidcells,it is necessaryfor the synthesisof hemoglobin.Two differentmechanismsexistfor the regulationof heme biosynthesisin the Iiver and the erythroidcells. Regulationin the liver : The first committed stepin hemebiosynthesis,catalysedby 6-amino- levulinate(ALA) synthase,is regulatory.Heme or itsoxidizedproducthemin (Fe3+)controlsthis enzyme activity by three mechanisms 1. Feedbackinhibition 2. Repressionof ALA synthatase 3. Inhibition of transport of ALA synthase from cytosolto mitochondria(thesiteof action). Effectof drugs on ALA synthaseactivity : The activity of ALA synthaseis markedly increased by the administrationof a largenumberof drugs e.g. phenobarbital, insecticides, carcinogensetc. This is expected since these compounds are mostly metabolized by a heme containing protein,cytochromePa56.On administrationof drugs,cellular levelsof heme are depleteddue to its increasedincorporationinto cytochrome Pa5s.The reducedhemeconcentrationincreases the synthesis(derepression)of ALA synthaseto meetthe cellulardemands.
  • Chapter 1O : HEMOGLOBINAND PORPHYRINS 271 cooH l (CHr), C-O + O-CoA Succinyl CoA cHr-NH, t- cooH Glyclne cr-Amino p-ketoadipate I CO,+-1 6-Arninolevulinatesynthase I + cooH I (?r,), ^-n | 8-Amlnolevullnate CH2NH2 (ALA) COOH COOH II 2H2o+-1o,HH,gh1Hl1"'" J OHCO COOH ll CH, (CHo), l'l-- c---c illl NH, I -I I I 4 PBG molecules Flg. 10.20 conld. next column Uroporphyrinogenlll I Uroporphyrinogen MP M P PM Coproporphyrinogenlll lCo *1 MV PM Protoporphyrlnogen lX MV PM Protoporphyrln lX I Fe2*--.1 FonocheJaiase J MV PM HEME M P FIg. 10.20: Biosynthesisot heme[A-Acetyl (- - COC ); P-P ropionyl (- CH2- CH2- COC );
  • 212 BIOCHEMISTRY SuccinylCoA+ Glycine | .. I D-Aminolevulinate HEMOGLOBIN I synthase t .,. +N-F... 6-Aminolevulinate(ALA) I - l^.- ... u.Jr,*''.. .1AlAdehydratase Fenp9hetatase . t='...nl +Uroporphyrinogen| + Lioht lroporitQn lll#-UroPot 3 | I Lioht I- Coproporphyrinlll Fig. 10.21: Summaryofhemesynthesisalongwithporphyilas (1-Acuteintermittentporphyia;2-Congenitalerythropoieticporphyria;3-Porphyria cutaneatarda;4-Hereditarycoptoporphyia;S-Variegateporphyria;6-Protoporphyria). Regulation in the erythroid cells : The enzymeALA synthasedoesnot appearto control the heme synthesis in the erythroid cells. Uroporphyrinogen synthase and ferrochelatse mostly regulateheme formation in these cells. Further, the cellular uptake of iron also influencesheme synthesis.lt is observedthat heme stimulatesglobin synthesis.This ensures that hemeand globin synthesisoccur in the right proportionto finally form hemoglobin. Porphyriasare the metabolic disorders of heme synthesis,characterizedby the increased excretion of porphyrins or porphyrin precurcors.Porphyriasare either inherited or acquired.They are broadly classifiedinto two categories 1. Erythropoietic: Enzymedeficiencyoccurs in the erythrocytes. 2. Hepatic : Enzymedefect lies in the liver. All the known porphyriasare inheritedas autosomal dominant disorders. Howere' congenital erythropoietic porphyria is a' exception,since it is autosomalrecessive.The different types of porphyrias are describec (Fig.l0.21, Table 10.3 l. Acute intermittent porphyria This disorderoccursdue to the deficiencyo- the enzyme uroporphyrinogen I synthase.Acute intermittent porphyria is characterized br increased excretion of porphobilinogen and 6-aminolevulinate.The urine gets darkenedon exposure to air due to the conversion ol porphobilinogento porphobilinand porphvrir The other characteristic features of acut€ intermittentporphyriaare as follows . lt is usuallyexpressedafterpubertyin human= . The symptoms include abdominal pai- vomiting and cardiovascularabnormalities The neuropsychiatricdistrubancesobserved- these patients are believed to be due :: reduced activity of tryptophan pyrrola=
  • Chapter 1O : HEMOGLOBINAND PORPHYRINS 213 Type of porphyria Enzvmedefect Characteristics Hepatic Acuteintermittentporphyria Porphyriacutaneatarda Hereditarycoproporphyria Variegateporphyria UroporphyrinogenI synthase Uroporphyrinogendecarboxylase Corpoporphyrinogenoxidase Protoporphyrinogenoxidase Abdominalpain,neuropsychiatric symFoms Photosensitivity Abdominalpain,photosensitivity, neuropsychiatricsymptoms Abdominalpain,photosensitivity, neuropsychiatricsymptoms Erythropoietic Congenitalerythropoieticporphyria Protoporphyria Uroporphyrinogenlll cosynthase Fenochelatase Photosensitivity,increasedhemolysis Photosensitivity (causedby depleted heme levels),resulting in the accumulation of tryptophan and 5-hydroxytryptamine. . The symptoms are more severe after administrationof drugs(e.g.barbiturates)that inducethe synthesisof cytochromePouo.This isdueto the increasedactivityof ALA synthase causingaccumulationof PBC and ALA. . Thesepatientsare not photosensitivesincethe enzymedefectoccurspri