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Biochemistry by satanarayana

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very helpful book of biochemistry

very helpful book of biochemistry

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  • 1. Dr, lJ, Satyan arayana F.l M . S c,.P h.D ., .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 An|D BCDCDT(S ALLTED lPf Ltd. No.1-E(1) "SHUBHAM PLAzA" (lst Floor) 83/I, BBLrRcrnrn MarN Roeo, Korrere 700010 (Ixora) k: i : (+9| -33) 653 -3844,2241-857 o F ax : (033)23 3 5 58-2127 e-mail : books cal.vsnl.net.in @
  • 2. Eiochemistrg First Published : March 1999 Reprinted: 1999 Revised Reprint : August 2000 Reprinted: 2OQO, 2001, 2QO2 Second Revised Edition : June 2002 Reprinted: 2003 Revised Reprint : 2004 Revised Reprint : 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 Arunabha Sen BOOKS AND ALLIED (P) Lro. 8/1 ChintamoniDas Lane, Kolkata 700009 Typesetter BOOKS AND ALLIED (P) Lro. 8/1 ChintamoniDas Lane, Kolkata 700009 Printer SWAPNA PRINTINGWORKS (P) Lro. 52 Raja Rammohan Roy Sarani, Kolkata 700009 Project Supervisor : Shyamal Bhattacharya t s B N B t - 8 ? !1 q -8 0 -t and Seventy{ive) only Price : Rs.575.00 lRupeesFivehundred US $12.00 only AuthorsSponsored Supported : & by INTERTINKS UIBFALAAUTFIOR-PUBTISHER (A"Pl D.No.: 48-16-10, Nagarjuna Nagar, Mahanadu Road, Vrjayawada-520008
  • 3. Prefaceto the Third Edition The response the first and the second to editionsof my book 'Biochemistry' (reprinted several times in just 6 years)from the students and teachers simply overwhelming. was flooded is I with highly appreciative lettersfrom all cornersof India and abroad! This givesme immensesatisfaction encouragemLnt this and in academic venture. I havecorresponded many biochernistry with teachers, inviting their comments and opinionsfor further improvingthe book. Most of them havebeenkind enoughto offer constructive suggestions. also visited I several colleges had personal and interaction with facultymembers and students. These exercises, spread over the past 6 years,have helped me to get direct feedback my book, besidesrealisingthe additional on requirements students. of I havegreat pleasure presentingthe third edition of my book with severalunique/novel in features, some high-lightsof which are listedbelow. . A thoroughrevisionand updatingof eachchapterwith latestadvances. Multicoloured illustrations a better understanding chemicalstructures for of and biochemical reactions. . Increase the font size of the text for more pleasant in and comfortable reading. o Incorporation a new Sectionon MolecularBiologyand Biotechnology. of . Addition of ten new chapters-human genomeproject,gene therapy,bioinformatics, free radicalsand antioxidants, tissueproteinsand body fluids,environmental genetics, biochemistry, immunologyetc. . An improvedorientationand treatmentof human biochemistry healthand disease. in . Additionof practicalbiochemistry and clinical biochemistry laboratory the appendix. in It is true that I represent selected group of individuals a authoringbooks,havingsometime at disposal, besides hard work, determination and dedication. consider I myselfan eternallearnerand a regularstudent of biochemistry. However, is beyond capability keeptrack of the evergrowing it my to advances biochemistry in due to the exponentialSrowth of the subject.And this makesme nervous,wheneverI think of revising the book. I honestlyadmit that I haveto depend mature readers subsequent on for editionsof this book. AN INVITATION TO READERS It is not all the time possible me to meetthe readers for individually and get their feedback, despite my ferventwish. Of course, do write to somepeople I personaliy seeking their opinions. However, wish to have I the comments suggestions eachoneof the readers my book.I sincerely and of of invite the readers feelfree to and write to me expressing their frank opinions,critical comments and constructive suggestions. DT. U. SATYANARAYANA tr l
  • 4. I owe a deepdebt of gratitude to my parents,the late Sri U. VenkataSubbaiah, and Smt. Vajramma,for cultivatingin me the habit of earlyrising.The writing of this bookwould neverhavebeenpossible without this healthyhabit. I am gratefulto Dr. B. S. Narasinga (formerDirector,NationalInstituteof Nutrition, Rao Hyderabad) disciplining my professional for life, and to my eldestbrother Dr. U. Gudaru(former Professor of PowerSystems, Walchand College Engineering, of Sangli)for discipliningmy personal life. (MBBS)deserves specialplace in this book. He made a significant My elder son, U. Chakrapani a contribution at everystageof its preparation-writing, verification,proof-reading what not. I had the rare and privilege teaching son as he happened be a studentof our college. of my to And a major part of this bookwas written while he waslearningbiochemistry. Thus,he wasthe first person learnthe subjectof biochemistry to from my handwrittenmanuscript.The student-teacher relation (rather than the father-son)has helpedme in receivinSl constant feedback from him and restructure the book in a way an undergraduate student would expecta biochemistry textbookto be. Next,I thank Dr. G. Pitcheswara (former Professor Anatomy,SMC,Vijayawada) his constructive Rao of for criticism and advice,and Dr. B. Sivakumar(Director,NationalInstitute of Nutrition, Hyderabad) his for helpful sugi5lestions the microfigures.I am grateful to my nephew,Mr. U. SrinivasaRao,for helping me on in drawingsomefigures. Last but not least, I thank my wife Krishna Kumari and my younger son, Amrutpani, without whose cooperationand encouragement this book could never have been written. The manuscript was carefully nurtured like a new born babyand the book has now become full-pledged a memberof our family. ACKNOWLEDGEMENTS THE THIRD EDITION TO pen-friends students I am indebted a largenumberof friends, to and who helped to revise me and improve the qualityof this book.I haveindividually personally and thanked of them (whonumbera fewhundreds!). all I onceagainexpress gratitudeto them. my I thank my friend and colleague, Mr. M.S.T.JaganMohan, who has helped me with his frequent interactions improvethe book,and makeit more student-friendly.would like to placeon recordmy deep to I (M.D.)students, (Mrs.)U.B.VijayaLakshmiand Dr. (Mrs.)Vidya sense appreciation my post-graduate of to Dr. Desai Sripad, whoseperiodical academic interaction and feedback havecontributed the improvement the to of biomedicaVclinical aspects somechapters. acknowledge help of my friend,Dr. P. Ramanujam in I the (Reader in English,AndhraLoyolaCollege, for Vijayawada) his help and encouragement revisingthe book. in I expressmy gratitude to Mr. ArunabhaSen, Director, Books & Allied (P) Ltd. Kolkata, for his wholehearted supportand constantencouragement revisingthe book in multicolour,and taking all the in pains to bring it out to my satisfaction. thank Mr. ShyamalBhattacharya his excellentpage-making I for and graphics-work the book.I am indebted Mr. Prasenjit in to Halderfor the coverdesignof this book. I thank my wife, Krishna Kumari, and my younger son, Amrutpani, for their constant support and encouragement. am grateful to UppalaAuthor-Publisher I Interlinks, Vijayawada, sponsoringand for supporting to bring out this edition. me DT. U. SAIYANARAYANA Ii i i ]
  • 5. Biochemistry The term Biochemistry was introduced by Carl Neuberg in 1903. Biochemistrybroadly dealswith the in chemistrv of life and living processes. There is no exaggeration the statement,'Thescopeof biochemistrg agingand death,involvesbiochemistry. is as uastas lilb itself !' Everyaspect life-birth, growth, reproduction, of For that matter, everymovementof life is packed with hundredsof biochemicalreactions.Biochemistryis the from the factthat over This becomes evident mostrapidlydeveloping mostinnovative and in subject medicine. the years,the major share of Nobel Prizesearmarkedfor Medicineand Physiologyhas gone to researchers engaged biochemistry. ir: The discipline of biochemistryservesas a torch light to trace the intricate complexicitiesof biology, unravelling chemical research amplydemonstrated all living has that besides the mysteries life. Biochemical of is things are closelyrelatedat the molecularlevel.Thus biochemistry the subjectof unity in the diversified living kingdom. Advances biochemistryhavetremendous in impact on human welfare,and havelargelybenefitedmankind of in for and their living styles.Theseincludethe application biochernistry the laboratory the diagnosis of and from geneticengiineering, the diseases. products(insulin,interferon, the €rowth hormoneetc.)obtained possibleuse of genetherapy in the near future. 0rganization of the Book This texthook,comprising43 chapters, orgianized secl:ions the heirarchicalorder of in is into serren learninSbiochemistry. . SectionI dealswith the chemicalconstituents life-carbohydrates, lipids,proteinsand amino acids, of nucleicacidsand enzymes. plasmaproteins,hemoglobin . SectionII physiological and chemistryincludesdigestion and ahsorption, prophyrins, and biological oxidation. (carbohydrates, . SectionIII incorporates the metabolisms minerals) lipids,amino acids,nucleotides, all . Section [V covershormones,organ function tests,water,electrolyteand acid-base balance, tissueproteins and trodi'fluids,and nutrition. (DNA-replication, . Section V is exclusively recombination, devoted molecularbiologyand biotechnology to DNAandbiotechnology) ar"ln repair, transcription translation, and recombinant regulation geneexpression, of . Section VI gives relevant information on current topics such a^s human genomeproject, gene therapy, prostaglandins, bioirrtormatics, diabetes, cancer, AIDS etc. . Section VII deals with the basic aspectsfor learning and understandingbiochemistry (bioorganic genetics, immunology). chenristry', chemistrytools of biochemistry, hiophysical to Each chapter in this book is carefully crafted with colour illustrations, headingsand subheadings quick understanding. importantapplications biochemistry humanhealthand disease put are facilitate The to of together as biomedical/clinical concepts. Icons are used at appropriateplacesto serveas 'landmarks'. practical biochemistryand clinical The origins of biochemicalwords, confusables biochemistry, in biochemistrylaboratory,given in the appendixare novel features. The briok is so organized to equip the readers knowledge biochemistry. of as with a comprehensive Ii u]
  • 6. Gontents SECTION ONE of ChemicalConstituents Life 1 > Biomoleculesthecell and 3 2 > Carbohydrates 9 3 > Lioids 28 4 > Proteins amino and acids 43 acids 5 > Nucleic andnucleotides 69 6 > Enzymes 85 7 > Vitamins 176 TWO SECTION Physiological Biochemistry B > Digestion absorption and 165 9 > Plasma oroteins 182 10 > Hemoglobin porphyrins and 196 11 > Biologicaloxidation SECTION FIVE Molecular Biologyand Biotechnology 24 > DNA-replication, recombination and repair 523 25 > Transcriotiontranslation and 542 26 > Regulation of gene expression 566 27 b Recombinant andbiotechnology DNA 578 sEcTtcN Current 28 > 29 > 30 F 31 p 32 33 34 35 36 >' > b > l" 221 SECTION 37> 38> THBEE stx Topics genome project Human 619 Gene therapy 625 Bioinformatics 634 'lvletabolism (detoxification) of xenobiotics 638 Prostaglandins related and compounds644 Biological membranes transport 650 and Freeradicals antioxidants and 655 Environmental biochemistry 662 glucose Insulin, homeostasis, mellitus anddiabetes 669 Cancer 58s Acquired immunodeficiency (AIDS) syndrome 695 241 q3 > Metabolism of carbohydrates 244 *4 > Metabolism of lioids 285 acids Metabolism of amino F-, 16 > Int6gration of metabolism 330. 380 17 > Metabolism of nucleotides 387 metabolism 1B > Mineral 403 FOUR SECTION and Nutrition Clinical Biochemistrv 19 > Hormones function tests 20 > Organ 21 > Water, electrolyte and bqlance acid-base > Tissue proteins body fluids and 22 23 > Nutrition- SECTION SEVEN Basics Learn Biochemistrv to 39 > Introduction to bioorganic chemistry 40 > Overview biophysical of chemistry 41 > Tools biochemistrv of 42 > lmmunology 43 > Genetics 487 APPENDICES AnswersSelf-assessmenl to Exercises I Abbreviations in this used book' ll Greek alphabets lll Origins important words ol biochemical lV Common confusables in biochemistry V Practical biochemistry-principles Vl Clinical laboratory biochemistry 502 INEEX 427 453 468 703 708 71 9 732 73 7 745 751 756 tJt 760 764 77 0 77 3
  • 7. fi Protuinsand Amino acids 4: Nucleic acidsand Nucleotides 69
  • 8. BflomnoXeeutrssCelll aildths -l- hu living matter is composed of mainly six hydrogen, oxygenl I elements-carbon, nitrogen, phosphorus and sulfur. These elements togetherconstituteabout 90% of the dry weight of the human body. Severalother functionally important elementsare also found in the cells. These include Ca, K, Na, Cl, Mg, Fe, Cu, Co, l, Zn, F, Mo and Se. organic compounds.lt is believedthat man may contain about 100,000 different types of molecules although only a few of them have been characterized. Sornpiex *riomoleeules The organic compoundssuch as amino acids, nucleotidesand monosaccharides serve as the monomeric units or building blocks of complex biomolecules-proteins,nucleic acids (DNA and earbon-a unique element of life RNA) and polysaccharides,respectively.The Carbon is the most predominantand versatile important biomolecules(macromolecules) with elementof life. lt possesses unique propertyto a their respective building blocks and major form infinite number of compounds. This is functions are given in Table 1.1. As regards attributed to the ability of carbon to form stable lipids, it may be noted that they are not c ov ale n t b o n d s a n d C -C c h a i n s of unl i mi ted biopolymers in a strict sense, but majority of length. lt is estimated that about 90% of them contain fatty acids. compounds found in living system invariably contain carbon. Structural heirarehy off asn organisnl (proteins, The macromolecules Iipids, nucleic acids and polysaccharides) form supramolecular Life is composed of lifeless chemical assembl i es(e.g. membranes)w hi ch i n tu r n molecules. A single cell of the bacterium, organize into organelles,cells, tissues,organs Escherichia coli contains about 6.000 different and fi nal l y the w hol e organi sm. Ghemical molecules of li#e 3
  • 9. B IOC H E MIS TFIY Major functions Building block (repeatingunit) Biomolecule 1. Protein Amino acids and Fundamental ofstructure basis functions). function cell(static dynamic and of (DNA) acid 2. Deoxyribonucleic Deoxyribonucleotides Ribonucleotides o.l.! iF 9199 ry fl_eq_o_sitoryi{9l1llgt acid 3. Ribonucleic (RNA) (glucose) 4. Polysaccharide(glycogen) Monosaccharides glycerol Fatty acids, 5. Lipid Chem *c a ! c o m p o s i ti o n biosynthesis. required Essentially lorprotein to short form Storage ofenergy meet term demands. to long torm Storage ofenergy meet term of membranes. components demands; structural Prokaryotic o f ma n The chemical compositionof a normal man, weighing 65 kg, is given in Table 1.2. Water is the solvent of life and contributesto more than 60"h of the weight. This is followed by protein ( m os t lyin mu s c l e )a n d l i p i d (mo s tl yi n adi pose 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. and eukaryotic cells The cel l s of the l i vi ng ki ngdom may be divided into two categories 1. Prokaryotes(Creek : pro - before; karyon and possess l nucl eus)ack a w el l defi nednucl eus simple structure. These include the relatively various bacteria. 2. E ukaryotes(Greek: eu-true; karyona nucleus)possess well defined nucleusand are more complex in their structure and function. (ani mal sand pl ants)are The hi gher organi sms composedof eukaryoticcells. between A comparisonof the characteristics prokaryotesand eukaryotesis listed in Table 1.3. The concept of cell originated from the o a c ont r ibut i o n s f S c h l e i d e n n d S c h w a n n(1838). However, it was only after 1940, the complexitiesof cell structurewere exposed. Percent(7") Weight (kg) Water 6 1 .6 40 Protein 17.0 11 Lipid 1 3 .8 I '| 6 .1 4 Constituent Carbohydrate Minerals The human body is composedof about 1014 cells. There are about 250 types of specialized cel{s in- the human body'G.g. erythrocytes, nerve-cells, muscle cells, B cells of pancreas. A n eukaryoti ccel l i s general l y10 to 100 pm in diameter. A diagrammatic representation of a typical rat liver cell is depicted in Fi g.I.t. The pl ant cel l di ffersfrom an ani mal cel l by possessing rigid cell wall (mostlycomposedof a cellulose) and chloroplasts.The latter are the sitesof photosynthesis.
  • 10. AND THE CELL Chapter 1 : BIOMOLECULES Eukaryotic cell Prokaryotic cell Characteristic (generally pm) 1-10 Small 1. Size 2. Cell membrane 3. Sub-cellular organelles 4, Nucleus metabolism 5. Energy 6. Celldivision 7. Cytoplasm DNA Notwell defined; isfound histones absent are asnucleoid, of Mitochondria enzymes absent, metabolism to bound energy membrane fission nomitosis Usually and and 0rganelles cytoskeleton absent T he c e l l c o n s i s ts f w e l l d e fi n e d subcel l ul ar o organelles,enveloped by a plasma membrane. tissue By differential centrifugation of homogenate, it is possible to isolate each c ellular o rg a n e l l e i n a re l a ti v e l y p ure form (Refer Chapter 41). The distribution of major enzymes and metabolic pathways in different c ellular o rg a n e l l e s i s g i v e n i n th e chapter on enzymes (Refer Fig.6.6). The subcellular organelles are briefly describedin the following pages. (generally pm) Large 10-100 plasma flexible Cell envelopeda is by membrane Distinct organellesfound are (e.9. mitochondria, lysosomes) nucleus, Nucleus defined, iswell surrounded bya membrane: isassociated histones DNA with Enzymesenergy metabolism located ol are inmitochondria Mitosis Contains organelles cytoskeleton and (anetwork oftubules filaments) and N ucl eus N ucl eus i s the l argest cel l ul ar organel l e , surrounded bv a doubl e membrane nucl ear envel ope. The outer membrane i s conti nuous w i th the membranes endopl asmi c reti cul um . of At certain intervals, the two nuclear membranes have nuclear pores with a diameterof about 90 nm. These pores permit the free passage the of products synthesizedin the nucleus into the surroundi cytopl asm. ng Mitochondrion Plasma membrane Vacuole reticulum Roughendoplasmic Ribosomes Golgi apparatus Lysosome Peroxisome Cytoskeleton Cytosol pits Coated representation a nt liverell. of Ftg. 1.1: Diagrammatic
  • 11. B IOC H E MIS TF|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. assembly An of nucleosomesconstitutes chromatin fibres of (Creek'. chromosomes chroma - colour; soma bod y ). T h u s , a s i n g l e h u ma n c h romosome i s c om o o s e do f a b o u t a m i l l i o n n u cl eosomes. The number of chromosomes is a characteristic feature of the species. Humans have 46 chromosomes, compactlypacked in the nucleus. The nucleusof the eukaryoticcell containsa dense bodv known as nucleolus.lt is rich in RNA, p a rti c u l a rl y th e ri b o s o mal R N A w hi ch entersthe cytosol through nuclear pores. 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 parts of the cell to provide energy for the cel l ul arw ork. matri x contai nsa ci rcular The mi tochondri al double stranded DNA (mtDNA), RNA and ribosomes. Thus, the mitochondriaare equipped with an independent protein synthesizing that about 10% of the machinery.It is estimated mitochondrial oroteins are produced in the mi tochondri a. The ground material of the nucleus is often referredto as nucleoplasm. lt is rich in enzymes p o l y me ra s e s and R N A s uc h a s D N A polymerases. the surpriseof biochemists, To the enzy m e s o f g l y c o l y s i s ,c i tri c a ci d cycl e and hexose monophosphateshunt have also been detected in the nucleoplasm. The structureand functions of mitochondria closely resemble prokaryotic cells. lt is hypothesizedthat mitochondria have evolved from aerobic bacteria.Further,it is believedthat during evolution,the aerobicbacteriadeveloped a symbi oti c rel ati onshi p w i th pri mor dial anaerobiceukaryoticcells that ultimately led to the arrival of aerobic eukaryotes. Mitochondria Endoplasmic The mitochondria (Creek'. mitos - thread; chondros- granule) are the centres for the c ell u l a rre s p i ra ti o n n d e n e rg ymetabol i sm. a They are regarded as the power houses of the cell wit h v a ri a b l es i z e a n d s h a p e .Mi t ochondri aare r od -l i k e o r fi l a me n to u s b o d i e s , usual l v w i th dim e n s i o n s o f 1 .0 x 3 p m. A bout 2,0O0 mitochondria,occupying about 1/5thof the total c ell v o l u me , a re p re s e n ti n a ty p ical cel l . The network of membrane enclosed spaces that extends throughout the cytoplasm constitutes endoplasmicreticulum (ER).Some of these thread-like structures extend from the nuclear pores to the plasma membrane. reticulum A large portion of the ER is studded with which ribosomesto give a granularappearance is referred ro as rough endoplasmic reticulum. Ribosomes are the factories of protein biosynthesis. During the process of cell The mitochondriaare comoosedof a double rough ER is disruptedto form small membrane system. The outer membrane is fractionation, vesicles known as microsomes. It may be noted smooth and completely envelopsthe organelle. The inner membrane is folded to form cristae that microsomesas such do not occur in the (Latin- crests) which occupy a larger surface cel l . area. The internal chamber of mitochondria is The smooth endoplasmicreticulum does not referred to as matrix or mitosol. lt contain ribosomes. is involved in the synthesis phospholipids, sterols) of lipids (triacylglycerols, The componentsof electron transportchain of supplyingCa'?. and metabolism drugs,besides and oxidative phosphorylation (flavoprotein, c y t o c h ro m e sb , c 1 , C , a a n d a 3 and coupl i ng for the cel l ul arfuncti ons. factors) are buried in the inner mitochondrial Golgi apparats,r$ membrane. The matrix containsseveralenzvmes concerned with the energy metabolism of E ukaryoti c l s contai n a uni que cl ust erof cel c ar b o h y d ra te si,p i d sa n d a m i n o a c i ds(e.g., tri c membrane vesicles known as dictyosomes l ci
  • 12. AND THE CELL Chapter 1 : BIOMOLECULES whic h, in tu rn , c o n s ti tu teC o l g i a p p a ratus(or proteins Colgi complex).The newly synthesized are handed over to the Colgi apparatuswhich lipids or catalysethe addition of carbohydrates, sulfatemoietiesto the proteins.Thesechemical for modificationsare necessary the transportof proteinsacrossthe plasma membrane. The pH of the lysosomal matrix is more acidic (pH < 5) than the cytosol (pH-7) and this facilitates degradation different the of compounds. The lysosomal enzymes are responsible for maintaining the cellular compounds in a dynamic stafe, by their degradationand recycling. The degradedproducts leave the lysosomes, usually by diffusion, for reutilization by the cell. Certainproteinsand enzymesare enclosedin Sometimes,however, certain residual products, m em br ane v e s i c l e s o f C o l g i a p p a ra t us and rich in lipids and proteins, collectivelyknown as secreted from the cell after the appropriate Iipofuscinaccumulatein the cell. Lipofuscinis signals.The digestiveenzymes of pancreasare the age pigment or wear and tear pigmentwhich or oduc edi n th i s fa s h i o n . has been implicatedin ageingprocess. Colgi a p p a ra tu sa re a l s o i n v o l v e d i n the The digestive enzymesof cellular compounds membrane synthesis, particularly for the are confinedto the lvsosomes the best interest in f or m at ion o f i n tra c e l l u l a r o rg a n e l l e s (e.g. of the cell. Escape theseenzymesinto cytosol of peroxisomes, lysosomes). will destroythe functionalmacromolecules tne of cel l and resul t i n many compl i cati ons.The Lysosornes occurrence of several diseases(e.g. arthritis, allergicdisorders) beenpartly has Lysosomes are spherical vesicles enveloped musclediseases, to of enzymes. by a single membrane.Lysosomes are regarded attributed the release lysosomal as the digestivetract of the cell, since they are ac t iv ely in v o l v e d i n d i g e s ti o n o f cel l ul ar Feroxi somes s ubs t anc e s -n a m e l y p ro te i n s , l i p i d s , carboPeroxisomes, also known as microbodies, are enzymes hydratesand nucleic acids. Lysosomal si ngl e membranecel l ul ar organel l es. They are are categorizedas hydrolases.These include the spherical or oval in shape and contain the following enzymes(with substratein brackets) enzyme catalase.Catalaseprotects the cell from (glycogen) a-C lucosidase the toxic effectsof HrO, by converting it to HrO and Or. Peroxisomes are also involved in tne (proteins) Cathepsins oxidation of long chain fatty acids (> C,s),and (l Lipases ipids) synthesis plasmalogens of and glycolipids.Plants (R N A ) contain glyoxysomes, a specialized type of Ribonucleases BTOMED|eAL CLINICAL COIUCEPTS / of A liuing cell is a true representotiue life with its own organizotion and specialized lunctions. Accumulotion oJ lipofuscin, a pigment rich in lipids and proteins, in the cell has been 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.
  • 13. E } IOC H E MIS TF| Y per ox is o m e s , w h i c h glyoxylate pathway. a re i n v o l v e d i n the Peroxisome biogenesis disorders (PBDs), are a Broup of rare diseasesinvolving the enzyme activities of peroxisomes. The biochemical abnor m a l i ti e sa s s o c i a te dw i th P BD s i ncl uoe increasedlevels of very long chain fatty acids (C2aand C26) and decreasedconcentrations of plasmalogens. The most severeform of PBDs is Zellweger syndrome, a condition characterized by the absenceof functional peroxisomes. The v ic t im s o f th i s d i s e a s e a v d i e w i th i n one vear m after birth. {iytosol and cytoskeleton The cellular matrix is collectively referred to as cytosol. Cytosol is basically a compartment containing several enzymes/ metabolites and saltsin an aqueousgel like medium. More recent studies however, indicate that the cytoplasm actually contains a complex network of protein filaments, spread throughout, that constitutes cytoskeleton. The cytoplasmic filaments are of three types- microtubules, actin filaments and filaments.The filamentswhich are intermediate polymers of proteins are responsiblefor the structure,shape and organizationof the cell. IN TE GR A TIOI{ OF C E LLU LA R FU N C TION S The eukaryoticcells perform a wide rangeof to complex reactionsfunctions maintain tissues, and for the ul ti mate w el l -bei ng of the w hol e organi sm. For thi s purpose, the vari ous processes biochemicalreactions intracellular and Division of are tightly controlled and integrated. cel a cel l i nto tw o daughter l s i s good exampl eof seriesof the orderly occurrenceof an integrated cel l ul ar reacti ons. Apoptosisis the programmed cell death or cel l sui ci de. Thi s occurs w hen the cel l has ful fi l l ed i ts bi ol ogi calfuncti ons.A poptosi smay be regardedas a natural cell death and it differs from the cell death caused by injury due to radiation,anoxia etc. Programmed cell death is a highly regulatedprocess. 1. 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, monosaccharides and fotty acids, respectluely. 2 . The cell is the structurol and functional unit of life. The eukoryotic cell consisfsof well det'inedsubcellulor organelles,enueloped in a plasma membrane. 3. The nucleus contoins DNA, the repository ol genetic int'ormation.DNA, in association with proteins (histones), which, in turn, make up the chromosomes. forms nucleosomes The mitochondria qre the centresfor energy metobolism. They are the principal producers of ATP which is exported to all parts of the cell to ptouide energy lor cellular work. 5. Endoplosmic reticulum (ER) ts the network of membrane enclosed spoces that 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 synthesized proteins are handed ouer for t'urther processing ond export. 6. Lysosomes are the digestiue bodies ol the cell, actiuely involued in the degradotion of cellular compounds. Peroxisomescontoln the enzyme catalose that 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. 7. The eukaryoticcellsperform a wide rangeof complex lunctionsin a well coordinatedand integrated fashion. Apoptosis is the processol programmed cell death or cell suicide.
  • 14. are 1^ arbohydrates the most abundant organic - m o l e c u l e s i n n a tu re . T h e y a re pri mari l y composedof the elementscarbon, hydrogen and oxygen.The name carbohydrateliterally means 'hydratesof carbon'. Some of the carbohydrates pos s es s e e mp i ri c a l fo rmu l a (C .H 2O)n here th w n 3 3, satisfying that these carbohydrates are in fact carbon hydrates. However,there are several non-carbohydrate compounds (e.g. acetic acid, C2HaO2; lactic acid, C3H6O3) which also appear as hydrates of carbon. Further, some of the genuine carbohydrates (e.g. rhamnohexose, C6H12O5i deoxyribose, C5H16Oa) not satisfy do the generalformula.Hence carbohydrates cannot be always consideredas hydratesof carbon. Carbohydrates may be defined as polyhydroxyaldehydes or ketones or compounds which produce them on hydrolysis. The term ' s ugar ' i s a p p l i e d to c a rb o h y d ra te s ubl e i n sol water and sweet to taste. #-ur*c;tEerEs earbohydrates of participatein a wide range of Carbohydrates f unc t io n s 1. They are the most abundantdietarysource of energy (a Cal/S)for all organisms. 2. Carbohydratesare precursors for many organic compounds (fats,amino acids). (as 3. Carbohydrates glycoproteins and glycolipids) participate in the structure of cell membraneand cel l ul ar functi onssuch as cell growth, adhesionand fertilization. 4. They are structuralcomponentsof many organi sms. Thesei ncl udethe fi ber (cel l ul ose) f o plants,exoskeleton some insectsand the cell of w al l of mi croorgani sms. 5. Carbohydrates also serve as the storage form of energy(glycogen) meet the immediate to energy demandsof the body. C LA S S IFIC A TION OF GARBOHYDRATES Carbohydrates are often referred to as saccharides (Greek: sakcharon-sugar). They are broadly classifiedinto three major groupsmonosaccharides, oligosaccharides and polysaccharides. This categorization is based on
  • 15. t0 B IOC H E MIS TR Y (empirical formula) Monosaccharides AIdose (CgHoOg) Trioses (C+HoO+) Telroses (CsHroOs) Pentoses (CoHrzOo) Hexoses (CzHr+Oz) Heptoses Glyceraldehyde Erythrose Ribose Glucose Glucoheptose Ketose Dihydroxyacetone Erythrulose Ribulose Fructose Sedoheptulose the number of sugar units. Mono- and oligosaccharides are sweet to taste, crystalline in characterand soluble in water, hence thev are commonly known as sugars. liberatedon hydrolysis.Basedon the number of monosaccharide units present, the oligosaccharides are further subdivided to disaccharides,trisaccharides etc. FJtonosaccharides P ol ysace hari des (Greek : mono-one)are the Monosaccharides group of carbohydrates simplest and are often referred to as simple sugars. They have the generalformula Cn(H20)n,and they cannot be further hydrolysed. The monosaccharides are divided into different categories,based on the functionalgroup and the numberof carbonatoms (Creek:poly-many)are polyPolysacchari6ls mers of mondficcharide units with high molecul ar w ei ght (up to a mi l l i on).They are usual l y and form colloids with tasteless(non-sugars) are of two typeswater. The polysaccharides homopolysaccharides and heteropolysaccharides. Aldoses : When the functional group in IH monosaccharides an aldehyde l-C:oi, is ,h"u are known as aldoses e.g. glyceraldehyde, gluc os e. is Stereoisomerism an importantcharacterof the monosaccharides. Stereoisomers are Ketoses When the functionalgroup is a keto : compounds that have the same structural lt formulae but differ in their spatialconfiguration. -C:O.l group, they are referred to as ketoses e.g. dihydroxyacetone, fructose. A carbon is said to be asymmetric when it is attached to four different atoms or groups. Ihe Based on the number of carbon atoms, the monosaccharides are regarded as trioses (3C), number of asymmetric carbon atoms (n) tetroses (4C), pentoses (5C), hexoses (6C) and determines the possible isomers of a given (7C).These heptoses terms along with functional compound w hi ch i s equal to 2n. C l ucose groupsare usedwhile naming monosaccharides. contains4 asymmetriccarbons,and thus has 16 For instance, glucose is an aldohexose while tsomers. fructose is a ketohexose(Table 2,1). Glyeeraldehyde T he c om mo n m o n o s a c c h a ri d e s d d i sacchaan -tfu e ref erqlrt*e cff rb$hyd$'er'&€3 rides of biological importanceare given in the (triose)is the simplestmonoClyceraldehyde Table 2.2. saccharide with one asymmetriccarbon atom. lt S S lgos ac c h a ri d e s and existsas two stereoisomers has been chosen (Creek: oligo-few) contain as the referencecarbohydrateio representthe Oligosaccharides 2- 1O m ono s a c c h a ri d em o l e c u l e s w h i ch are structureof all other carbohvdrates.
  • 16. Ghapter 2 : CARBOHYDRATES 11 Trioses in Glyceraldehyde Found cells phosphate as Dihydroxyacetone Found cells phosphate in as Tetroses D-Erythrose i i i Glyceraldehyde 3-phosphate intermediate isan inglycolysis ttst -pnosphate intermediate isan inglycolysis ----t - -- - --.. -.. -. --... -- - i Widespread Pentoses D-Ribose asa of I Widespread constituent I RNA nucleotides and D-Deoxyribose i Asa constituent ofDNA D-Ribulose during : Produced metabolism D-Xylose i i i i L-Xylulose D-Lyxose Asa constituent ofglycoproteins gums ano ls anintermediate acid inuronic pathway Heart muscle i --. --. -- --.. ---.. -.. -. --. Hexoses D-Glucose D-Galactose D-Mannose D-Fructose Asa constituent ol polysaccharides (starch, glycogen, cellulose) and (maltose, disaccharides lactose, Also in sucrose). found fruits Asa constituent oflactose (milk sugar) polysaccharides Found plant in glycoproteins and animal Fruits honey, a constituent and as and ofsucrose inulin Heptoses in D-SedoheptuloseFound olants Disaccharides Sucrose Lactose Maltose Occurrence Asa constituent ofcane sugar and pineapple beet sugar, Milk sugar Productstarch of hydrolysis, in seeds occurs germinating For structureRNA nucleotide the of and (ATP, coenzymes NAD+, NADP+) For structureDNA the ol Itisanimportant metabolite inhexose monophosphate shunt Involved functionglycoproteins inthe of pentosuria Excreted inurine essenlial in Asa constituent ol lvxollavinheart of muscle 'sugar oflife; The fuel' excreted inurine in diabetes. Structural ofcelluloseplants unit in Converted toglucose, leads failure to galactosemia For structurepolysaccharides the of Itsphosphates intermediates are ofglycolysis Its7-phosphate intermediatehexose isan in monophosphate andin photosynthesis shunt, Biochemical importance Most commonly table used sugar supplying calories Exclusive carbohydrate tobreast source fed (lactose infants. Lactase deficiency intolerance) leads dianhea flatulence to and Animportant intermediate digestion inlhe of starch
  • 17. E}IOCHEMISTFIY 12 H-C:O I H-C-OH cH2oH D-Glyceraldehyde H-C:O I H-C-OH I HO-C-H I H-C-OH I H-Q-OH I cHzoH D-Glucose H-C:O HO-C-H rotatethe plane to compoundsthat respectively of polarized light to the right or to the left. cH2oH An optical isomer may be designated as D(+), D(-), L(+) and L(-) based on its structural L-Glyceraldehyde relation with glyceraldehyde.lt may be noted of that the D- and L-configurations sugarsare H-C:O I primarily based on structure of the HO-C-H glyceraldehyde, the optical activities however, H-C-OH may be different. I HO-C-H HO-C-H cH2oH L-Glucose Fig.2.1 : DandL- forms of glucose compared with D and L- glyceraldehydes (the reference carbohydrate). Racemic mixture : lf D- and L-isomersare present in equal concentration,it is known as mi racemi cmi xtureor D L mi xture.R acemi c xture does not exhibit any optical activity, since the dextro- and levorotatorv activities cancel each other. Configuration D" and L-isomers The D and L isomers are mirror images of each other. The spatial orientation of -H and -OH groups on the carbon atom (Cs for glucose)that is adjacentto the terminal primary alcohol carbon determineswhether the sugar is D- or L-isomer.lf the -OH group is on the right side, the sugar is of D-series, and if on the left side, it belongs to L-series.The structuresof D- and L-glucosebased on the referencemonosaccharide, D- and L-glyceraldehyde (glycerose) are depicted in Fig.2.1. of D-aldoses The configuration of possible D-aldoses starting from D-glyceraldehydeis depicted in Fig.2.2. This is a representation of Killianithe chain length by Fischersynthesis, increasing of an aldose, by one carbon at a time. Thus, (3C),aldotetroses (4C), with an aldotriose starting aldopentoses (5C) and aldohexoses(6C) are glucose, mannose formed. Of the 8 aldohexoses, and galactose are the most familiar. Among these, D -gl ucose i s the onl y al dose monothat predominantlyoccurs in nature. saccharide Gonfiguration of D-ketoses It may be noted that the naturallyoccurring (triose), there Startingfrom dihydroxyacetone monosaccharides the mammalian tissuesare in physiologicallr which are are five keto-sugars mostlyof D-configuration. enzymemachinery The are given in Fig,2.3 important.Their structures of cells is specific to metabolise D-series of monosaccharides. Epimers fn the medical practice, the term dextrose is used for glucose in solution. This is because of the dextrorotatory nature of glucose. ff two monosaccharides differ from eacother in their configuration around a singk specificcarbon (other than anomeric) atom. L*ei are referred to as epimers to each orher '.Fig,21 Optlcal activity of sugars For instance, glucose and galactose are efilwl with regard to carbon 4 (Ca-epimers -^:i 's Optical activity is a characteristicfeature of they differ in the arrangementof -OH g.'ELcr compounds with asymmetric carbon atom. Clucose and mannose are epi-'e--' q drl When a beam of polarized light is passed Ca. regardto carbon 2 (C2-epimers). througha solutionof an optical isomer,it will be of rotated either to the right or left. The term The interconversion epimers e I r::r'e dextrorotatory (+) and levorotatory (-) are used to galactose and vice versai s i- - -^,' a*
  • 18. Ghapter 2 : CABBOHYDFATES 13 cHo I HCOH I cH2oH 'l Aldotriose (3c) t- Aldotetroses (4c) D-Erythrose D.Threooe cHo cHo H C OH H OC H I HCOH cHo HCOH cH2oH D-Arabinose HCOH I HCOH cH2oH D-Ribose cHo I H C OH I HOCH I HCOH cH2oH D-Xylose I HOCH Aldo I HOCH I toses ) HCOH I cH2oH D-Lyxoee I / JT cHo tl HCOH HCOH tl HCOH tl HCOH ll cH2oH D-Allose / / cHo HOCH HCOH JT cHo HCOH rl cHo HOCH HOCH HOCH tl HCOH HCOH HCOH tl HCOH HCOH HCOH ll cH2oH cH2oH cH2oH D-Altrose D-Glucose D-Mannose */ + cHo cHo I H C OH I HCOH I HOCH I HCOH cH2oH D-Gulose cHo I HCOH I HOCH I HCOH I HOCH I HOCH HoCH noCH HCOH I cH2oH D-ldose cHo H OC H I AldoHOCH hexoses tl HCOH tt cHzoH (6c) HCOH cHzoH D-Galactose D-Talose Fig.2.2 : Thestructuralrelationship betweenD-aldoses projection. shownin Fischer (Theconfiguration aroundC2(ed) distinguishes membersof eachpair). the epimerization, and a group of enzymesnamely-epimerases catalyse this reaction. The term diastereomersis used to represent the sfereoisomers that are not mirror images of one another. E nanti o m e rs Enantiomers are a special type of stereoisomers that are mirror images of each other. The two members are designatedas D- and L-sugars.Enantiomersof glucose are depicted in Fig.2.5. For a better understanding of glucose structure, let us consider the formation of hemiacetals and hemiketals, respectively Majority of the sugarsin the higher animals producedwhen an aldehydeor a ketone reacts (including man) are of D-type (Fig.2.5'1. w i th al cohol .
  • 19. E } IOC H E MIS TR Y 14 cH2oH cH2oH I cH2oH ?H2oH C :O cH20H I I C:O HCOH I I cH2oH cH2oH Dlhydroxyacetone D-Xylulose HOCH I HCOH C :O C :O I HOCH HCOH I HCOH I cH2oH HCOH I cH2oH D-Ribulose D-Fructose 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- Anomers-nrutarotation Rr- The a and p cyclic forms of D-glucose are known as anomers. Thev differ from each other Alcohol Aldefry<b Hemiacetal in the configurationonly around C1 known as The hydroxyl group of monosaccharides can anomericcarbon (hemiacetal carbon).In caseof react with its own aldehyde or keto functional o anomer, the -OH group hel d by anomeri c group to form hemiacetaland hemiketal.Thus, carbon is on the opposite side of the group the aldehydegroup of glucoseat C1 reactswith -CH2OH of sugar ring. The reverseis true for alcohol group at C5 to form two types of cyclic B-anomer. The anomersdiffer in certain physical hemiacetals namely a and B, as depicted in and chemical properties. Fig.2.6. The configuration of glucose is Mutarotation : The a and p anomers of conveniently represented either by Fischer glucose have different optical rotations. The formulae or by Haworth projectionformulae. specific optical rotation of a freshly prepared glucose(c anomer)solution in water is +112.2o Fyranose and furanose structures w hi ch gradual l y changes and attai ns an Haworth projectionformulae are depicted by equi l i bri umw i th a constantval ue of + 52.7" . l n ring pyranose(basedon pyran) the presenceof alkali, the decreasein optical a six-membered five-membered ring furanose (based on rotation is rapid. The optical rotation of or a furan).The cyclic forms of glucoseare known as p-glucose is +18.7o. Mutarotation is defined as a-D-glucopyranose and c-D-glucofuranose the change in the specific optical rotation (Fig.2.V. representing the interconversion of u and p LJ 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 H-C=O I I H-C-OH HO-C-H I I HO-C-H HO-C-H I I H .C -O H H-C-OH I I H-C-OH H-C-OH I I CHzOH cH2oH D-Glucose D-Mannose H O= C H O_C - H I H-C- Cl-i I H O-C -H I HO-C- Fl I H-C- ii I OH Fig.2,4: Structuresof epimers (glucose and galactose are Co-epimerswhile glucose and mannose are C2-epimers). L-Glucose H I C :O I f{ c-oH I H C -C -H i-l- c-oH H -C -OH I t"1-c-H HO D-Glucose (mirrorimages)of glucose. H9.2.5 : Enantiomers
  • 20. t5 Ghapter 2 : CARB 1 H -C :O I H -C -OH I H O-C -H I H -C -OH tc H -C -OH I cH2oH D-Glucose form) (aldehYde I cH20H o'D'Glucose (+ 112.2" iHron ftD-Glucose (+ 18.7-) cH20H (B) l/A fil H6?H H6?H o-D-GlucoPYranose H OH D-Glucose form,acYclic) (aldehyde H OH FD-GlucoPYranose the p 17oopen chai n form. l n aqueoussol uti on' 'i, D'glucose to an equilihrium mixture' forms of more predominant due to its stable forrn Mutarotationdepicted in Fig' 2'6, is summartzeo glucoseare conformation.The cr and p forms of below. occurs through a linear which interconvertible # mixture B-D-Clucose form. The latter, as such, is present in a" # cx-D-Clucose Equilibrium + 18.7" quanti tY . + 5 2 .7 " i nsi gni fi cant + 11 2 .2 " Mutarotation of fructose z Frur' mutarotation. ln case or 63o/" exhibits T h e e q u i l i b ri u m m i x tu re c o ntai ns pyranose ring (six-memberqd' p-anomer and 36"/ocl-anomer of glucose with furanose ive-membered)'o' (f is attained.And fruqt' rotation of -92)2. (Specificoptical rotation tctl2p0) I he conv' to levor ':ut" cH20H t- H-C-OFi OH HOH cr-D-GlucoPYranose HOH uranose cr-D-Glucof of Fig.2.7 : Structurc glucose-pyranose torms' and furanose on i s kn, anome' i n al kal i r W hen gt. severalhours, :;r'
  • 21. chapter 2 : CAFIBoHYDFATES 15 1 H -C = C ) I H-C-OH I HO-C-H I H-C-OH l5 H-C-OH I cH2oH cH2oH D-Glucose (aldehyde form) cr-D-Glucose (+ 112.2") cH2oH 20H H H OH o-D-Glucopyranose H OH D-Glucose (aldehyde form,acyclic) pD-Glucopyranose Fig. 2.6 : Mutarotation of glucose representing a and p anomers (A) Fischer projections (B) Haworth projections. forms of D-glucose to an equilibrium mixture. 17oopen chain form. In aqueoussolution,the p Mutarotationdepicted in Fi9.2.6, is summarized form is more predominant due to its stable conformation.The s and p forms of glucoseare below. interconvertible which occurs through a linear s-D-Clucose Equilibrium mircture p-D-Glucose # # form. The latter, as such, is present in an + 112.2" + 52.7" + 18.7o quanti ty. i nsi gni fi cant (Specificoptical rotation talf;) Mutarotation of fructose : Fructose also exhibits mutarotation. ln case of fructose, the T h e e q u i l i b ri u m m i x tu re contai ns 63" /" is p-anomer and 36h cl-anomerof glucose with pyranose ring (six-membered) converted to furanose(five-membered) ring,till an equilibrium is attained.And fructose has a specific optical rotation of -92" at equilibrium. The conversion of dextrorotatory (+) sucrose to levorotatory fructose is explained under inversionof sucrose(see later in this chapter). REACTIONS OF MONOSACCHARIDES Tautomerization cr-D-Glucopyranose c-D-Glucofuranose Fig.2.7 : Structureof glucose-pyranose and furanose forms. or enolization The processof shiftinga hydrogenatom from one carbon atom to another to produce enediols is known as tautomerization. Sugarspossessing anomeric carbon atom undergo tautomerization i n al kal i nesol uti ons. When glucose is kept in alkaline solution for severalhours,it undergoes isomerization form to
  • 22. 16 BIOCHEMISTFIY H n-C-ot H-C:O I H- -OH ( HO-( 2H2O+ CueO {- HO-( R Enediol (common) t 2Cu(OH) It may be noted that the reducing property of of sugars cannot help for a specificidentification any one sugar,since it is a general reaction. 0xida*iern Depending on the oxidizing agent used, the terminal aldehyde (or keto) or the terminal Fig.2.8 : Formation a commonenediolfrom of alcohol or both the groupsmay be oxidized. For glucose,fructoseand mannose :PI:Is?Iboncolnmonstnf tar:?l,l instance,considerglucose : :!lo.t|tfr {fr,f,o,F|F|lPffi ,ft D-fructose and D-mannose. This reactionknown 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. 1. Oxidation of aldehyde group (CHO ------> COOH) resultsin the formationof gluconic acid. 2. Oxi dati on of termi nal al cohol grou p (CH2OH ------+ COOH) leadsto the production of gl ucuroni caci d. R edueti on The enediolsare highly reactive, hencesugars When treated with reducing agents such as in alkaline solution are powerful reducing sodium amalgam,the aldehydeor keto group of agents. monosaccharideis reduced to corresponding alcohol, as indicated by the generalformula : r!s.l FeF tlsF Fr'.lg ft+r,.lule H The sugarsare classifiedas reducingor nonH-C:O H-C-Ol-t reducing.The reducing property is attributedto I RR the free aldehyde or keto group of anomeric carbon. ln the laboratory, many testsare employed to identify the reducing action of sugars. These incf ude Benedict's test, Fehling's test, Barfoed's tesf etc. The reduction is much more efficient in t he a l k a l i n e me d i u m th a n i n the aci d m ediu m. The enediolforms (explained above)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. The important monosaccharidesand their alcohols are given below. corresponding D -Gl ucose D-Galactose------+ D-Dulcitol D-Mannose ------+ D-Mannitol D-Mannitol+ D-Sorbitol D-Fructose --) D-Ribitol D-Ribose -+ Sorbitol and dulcitol when accumulate in tissues in large amounts cause strong osmotic effects feading to swelling of cells, and certain pathological conditions.e.g. cataract,peripheral neuropathy,nephropathy.Mannitol is useful to reduce intracranialtension bv forced diuresis.
  • 23. 17 Ghapter 2 : CAFIBOHYDRATES H-C:O I H-C--O I H-C-OH I HO - C -H I H-C-OH I H-C-OH Formation of esters The al cohol i c groups of monosaccharides may be esterified by non-enzymatic or enzymatic reactions. Esterificationof carbohydrate with phosphoric acid is a common reaction in metabolism. Glucose 6-phosphate cH20H and glucose 1-phosphateare good examples. Hydrorymethyl furfural ATP donates the phosphate moiety in ester formation. I cH2oH D-Glucose H-C:O H-C:O I H-C-OH tl l l H-C-OH I C----r HeSoo Conc. I H-C-OH CHrou rH '1 3H2o D-Ribose H-Q I H-C L U I H-d---l Furfural 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 carbohydrate reactswith a hydroxyl group of another carbohydrate or a nonDehydration carbohydrate (e.g. methyl alcohol, phenol, sulfuricacid, glycerol). The bond so formed is known as When treatedwith concentrated undergo dehydrationwith an glycosidic bond and the non-carbohydrate monosaccharides elimination of 3 water molecules.Thus hexoses moiety (when present)is referredto as aglycone. give give hydroxymethylfurfural while pentoses The monosaccharides are held together by furfural on dehydration (Fi9.2.9).These furfurals gl ycosi di c bonds to resul t i n di -, ol i go- or c an co n d e n s e w i th p h e n o l i c c ompounds (see later for structures). polysaccharides (a-naphthol)to form coloured products.This is the chemical basis of the popular Molisch test. they are In case of oligo- and polysaccharides, H-C=O to by first hydrolysed monosaccharides acid, and + HrN-NH-CuHu I _ H -C -OH this is followed by dehydration. Fig.2.9 : Dehydration of monosaccharides with concentrated H o. "SO Osazone formation Phenylhydrazine in acetic acid, when boiled with reducing sugars, forms osazones in a reaction summarizedin 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 difference is Thus m as k e db y b i n d i n g w i th p h e n y l h y drazi ne. glucose, fructose and mannose give the same osazones. type (needle-shaped) Reducingdisaccharides also give osazonesmaltose sunflower-shaped,and lactose powderpuff shaped. 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 (R rcprcsents Crto Crof glucose).
  • 24. t8 B IOC H E MIS TR Y Naming of glycosidic bond : The nomenclatureof glycosidic bonds is based on the Iinkagesbetween the carbon atoms and 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 named as 0(.1 4) glycosidicbond. The other glycosidic bonds are described in the structure of di- an d p o l y s a c c h a ri d e s . Physiologieally important glycosides 1 . G lu c o v a n i l l i n (v a n i l l i n -D -g l u c o si de) a is natural substance that impartsvanilla flavour. The ami no groups of ami no sugars are sometimes acetylated e.g. N-acetyl D-glucosamrne. N -A cetyl neurami ni c aci d (N A N A ) i s a and pyruvic acid. derivativeof N-acetylmannose It is an important constituentof glycoproteins and glycolipids.The term sialic acid is used to include NANA and its other derivatives. C ertai n anti bi oti cs contai n ami no sugars which may be involved in the antibiotic activity e.g. erythromycin. 5. Deoxysugars: These are the sugarsthat 2. Cardiac glycosides(steroidalglycosides) contain one oxygen less than that present in the : Digoxin and digitoxin contain the aglycone parent mol ecul e. The groups -C H OH and steroid and they stimulatemuscle contraction. -C H 2OH become -C H 2 and -C H 3 due to the is of 3. Streptomycin, an antibiotic used in the absence oxygen. D-2-Deoxyribose the most important deoxysugar since it is a structural treatmentof tuberculosis a glycoside. is constituentof DNA (in contrastto D-ribose in 4. Ouabain inhibits Na+- K+ ATPase and R N A ). blocks the active transportof Na+. 6. L-Ascorbic acid (vitamin C) : This is a DERIVATIVES MONOSACCHARIDES OF water-soluble vitamin, the structure of which that of a monosaccharide. There are severalderivatives monosaccha- closely resembles of r ides , s o m e o f important which a re p h y s i ol ogi cal l y 1. Sugar acids : Oxidation of aldehyde or primaryalcohol group in monosaccharide results in sugar acids. Cluconic acid is produced from glucose by oxidation of aldehyde (C1 group) whereasglucuronicacid is formed when primary alc ohol g ro u p (C 6 )i s o x i d i z e d . 2. Sugar alcohols (polyols) : They are producedby reductionof aldoses ketoses. or For instance, sorbitol is formed from glucose and mannitol from mannose. 3. Alditols : The monosaccharides, on reduction,yield polyhydroxyalcohols,known as aldit ols . R i b i to l i s a c o n s ti tu e n t of fl avi n coenzymes; glycerol and myo-inositol are components lipids. Xylitol is a sweetener of used in s ugarl e s g u m s a n d c a n d i e s . s 4. Amino sugars : When one or more hydroxyl groups of the monosaccharides are replaced by amino groups, the products formed are amino sugars e.g. D-glucosamine, D-galactosamine. They are present as constituents of heteropolysaccharides. The structuresof selected monosaccharide derivativesare depicted in Fig.2.l1. di A mong the ol i gosacchari des, sacchari de s are the most common (Fig.2,l2). As is evident from the name, a disaccharideconsistsof two monosacchari de ts(si mi l ar di ssi mi l ar) d uni or hel together by a glycosidic hond. They are crystalline, water-soluble and sweetto taste.The disaccharides of two types are '1. Reducingdisaccharides with free aldehyde or keto group e.g. maltose, lactose. with no free 2. Non-reducing disaccharides aldehyde or keto group e.g. sucrose,trehalose. Maltose Maltose is composed of two a-D-glucose units held togetherby cl (1 -+ 4) glycosidicbond. The free aldehydegroup present C1 of second on glucoseanswersthe reducing reactions, besides
  • 25. Ghapter & : CAFIBOHYDRATES H -C :O I H-C-OH I H O -C -H I H-C-OH 19 cH2oH I H -C -OH I I H-C-OH I COOH D-Glucuronic acid cH2oH Glycerol H OH myo-lnositol H 3C -C --H N OH H D-2-Deoxyribose H NHz D-Glucosamine H OH N-Acetylneuraminic acid Fiq.2.11 : Structuresol monosaccharidederivatives(selectedexamples). the osazone formations (sunflower-shaped). and keto) are held togetherand protectedfrom Maltosecan be hydrolysedby dilute acid or the oxidative attacks. enzyme maltase to liberate two molecules of Sucrose is an important source of dietary cr-D-glucose. carbohydrate. lt is sweeter than most other (except common sugars fructose) namelyglucose, lactoseand maltose.Sucroseis employed as a agent in food industry.The intestinal Cellobioseis another disaccharide,identical sweetening enzyme-sucrase-hydrolysessucrose glucose to in structurewith maltose,exceptthat the former has p (1 -r 4 ) g l y c o s i d i cl i n k a g e .C e l l obi osei s and fructosewhich are absorbed. f or m ed d u ri n g th e h y d ro l y s i s f c e l l ul ose. o F-aetsse Suoroee Lactose is more commonlv known as milk ln isomaltose,the glucose units are held togetherby o (1 --+6) glycosidic linkage. (cane sugar) the sugarof commerce, is Sucrose mostly producedby sugarcane and sugar beets. Sucrose is made up of a-D-glucose and pD-fructose.The two monosaccharides held are together a glycosidicbond (a1 -+ B2),between by Cj of c-glucose and C2 of B-fructose.The reducing groups of glucose and fructose are i inv olv e di n g l y c o s i d i cb o n d , h e n c e s ucrose s a non-reducing sugar, and it cannot form osazones. sugarsi nce i t i s the di sacchari de found i n mi l k. Lactose is composed ol p-D-galactoseand B-Dglucoseheld together by 0 (1 -r a) glycosidic bond. The anomericcarbonof C1 glucoseis free, hence lactose exhibits reducing propertiesand (powder-puff hedgehog formsosazones or shape). Lactose of milk is the most important carbohydrate the nutritionof young mammals. in It is hydrolysedby the intestinalenzyme lactase Sucroseis the major carbohydrateproduced to glucoseand galactose. in photosynthesis. lt is transported into the storageorgans of plants (such as roots, tubers lnversion ef suerose and s ee d s ). c ro s es th e mo s t a b u n dantamong Su i Sucrose,as such is dextrorotatory(+66.5o). t he natu ra l l y o c c u rri n g s u g a rs .l t h as di sti nct But, r,r,hen hydrolysed, sucrose becomes advantages over other sugarsas a storageand levorotatory(-28.2"). The processof change in transoort form. This is due to the fact that in optical rotation from dextrorotatory (+) to sucrose, both the functional groups (aldehyde levorotatory(-) is referredto as inversion.The
  • 26. BIOCHEMISTF|Y (or consistof Polysaccharides simply glycans) repeat units of monosaccharides or their derivatives,held together by glycosidic bonds. They are primarilyconcernedwith two important and storageof energy. functions-structural, Polysaccharides are linear as well as branched polymers. This is in contrast to structure proteinsand nucleic acids which are of only linear polymers. The occurrence of is branches in polysaccharides due to the fact that glycosidic linkagescan be formed at any one of the hydroxylBroupsof a monosaccharide. H OH Glucose Fructose Sucrose (a-D-glucosyl --+ p-D-fructose) (1 2) Polysaccharides are of two types which on hydrolysis 1. Homopolysaccharides yield only a singletype of monosaccharide. They are named based on the nature of the unit. Thus,glucans are polymers monosaccharide of glucose whereas fructosans are polymers of fructose. on 2. Heteropofysaccharides hydrolysisyield or a mixture of a few monosaccharides their derivatives. Galactose Lactose (p-D-galactosyl-+ a) p-D-glucose) (1 $tarch Fig. 2.12 : Structures of disaccharides -maltose, sucrose and lactose. Starch is the carbohydrate reserve of plants which is the most important dietary source for hi gher ani mal s,i ncl udi ngman. H i gh contentof hydrolysed mixture of sucrose, containing starchis found in cereals,roots,tubers,vegetables gfucose and fructose, is known as invert sugar. etc. Starch is a homopolymer composed of The processof inversion is explained below. D-glucose units held by a-glycosidic bonds. lt is known as glucosan or glucan. Hydrolysis of sucrose by the enzyme sucrase (invertasd or dilute acid liberates one molecure Starch consists of two polysaccharide each of glucose and fructose. ft is postulatedthat components-water soluble amylose (15-20o/ol sucrose (dextro) is first split into a-D- and a water insoluble amylopectin (80-85%). glucopyranose(+52.5") and p-D-fructofuranose, Chemically, amylose is a long unbranched both being dextrorotatory. However, p-D- chain with 200-1,00OD-glucoseunits held by c fructofuranose lessstableand immediately gets (1 + 4) gl ycosi di cl i nkages. myl opecti n, the is on A converted to p-D-fructopyranose which is other hand, is a branchedchain with a (1 --r 6t (-92"). The overall effect is gl ycosi di cbonds at the branchi ngpoi nts and c stronglylevorotatory that dextro sucrose (+66.5") on inversion is (1 -; 4) linkages everywhere else (Fig.2.13). converted to levo form (28.2'. A myl opecti n mol ecul e contai ni ng a few
  • 27. 21 ChapteF 2 : CARBOHYDFATES D-Glucose D-Glucose o-Amylose +- 6nu vt (1-* 6) Branch t2 Main chain Lg Amylopectin thousand glucose units looks like a branched saccharideeasily soluble in water. Inulin is not utilized by the body. lt is used for assessing tree (20-30 glucose units per branch). amylase kidney function through measurement of Starches are hydrolysed by (pancreaticor salivary)to liberatedextrins,and glomerular filtration rate (GFR). finally maltoseand glucose units. Amylase acts Gl ycogen specificallyon a (1 -+ 4) glycosidic bonds. Clycogen is the carbohydrate reserve in animals, hence often referredro as animal starch. Dextrins Dextrins are the breakdown products of It is present in high concentration in liver, starch by the enzyme amylase or dilute acids. followed by muscle,brain etc. Clycogenis also chlorophyll Starch is sequentially hydrolysed through found in plants that do not possess (e.9. yeast,fungi). different dextrins and, finally, to maltose and (identifiedby glucose.The various intermediates The structureof glycogen is similar to that of iodine colouration) are soluble starch (blue), amylopectin with more number of branches. amylodextrin (violet), erythrodextrin (red) and Glucoseis the repeatingunit in glycogenjoined achrodextrin (no colour). togetherby u (1 + 4) glycosidic bonds, and a (1 + 6) gl ycosi di c bonds at branchi ng poi nt s I nulin (Fi9.2.1Q. The molecularweight (up to 1 x 108) fnulin is a polymer of fructosei.e., fructosan. and the number of glucose units (up to 25,000) I t oc c u rs i n d a h l i a b u l b s , g a rl i c ,o n i on etc. l t i s vary in glycogendependingon the source from a low m o l e c u l a r w e i g h t (a ro u n d 5 ,000) pol y- which glycogen is obtained.
  • 28. B IOC H E MIS TR Y 22 decreasing the absorption of glucose and from the intestine, besidesincreasing cholesterol (For details, Chapter 23) the bulk of feces. Ghitin Chitin is composed of N-acetyl Dglucosamineunits held together by F (1 -+ a) pol gl ycosi di c bonds.l t i s a structural ysaccha r ide found in the exoskeleton of some invertebrates e.g. insects,crustaceans. T N T Ot (B) 9H2OH y'-O., (+ -o-J, J uqt, , F--o. i) - r+ , . / ' o-- ' L-/ CH2oH ,r4-Or ,L^_K. " --l are composed of When the polysaccharides they or differenttypes of sugars their derivatives, or are referred to as heteropolvsaccharides heteroglycans. X^_ (A) Fiq.2.14: Structure glycogen General of structure (B) Enlarged a branchpoint. at Cellulose i Ce l l u l o s e c c u rse x c l u s i v e l yn p l antsand i t i s o the most abundant organic substancein plant k ingd o m . l t i s a p re d o m i n a n t c onsti tuentof plant c e l l w a l l . C e l l u l o s e i s to ta l ly absent i n anim a l b o d y . Cellulose is composed of p-D-glucose units linked by 9 0 -+ 4) glycosidic bonds (Fi9.2.1fl. Cellu l o s e c a n n o t b e d i g e s te d b y mammal sincluding man-due to lack of the enzyme that cleavesB-glycosidic bonds (a amylasebreakscr bonds only). Certain ruminantsand herbivorous n anim a l s o n ta i nm i c ro o rg a n i s mi s the gut w hi ch c produce enzymes that can cleave p-glycosidic bonds . H y d ro l y s i s o f c e l l u l o s e yi el ds a disaccharide cellobiose, followed by P-Dglucose. Cellulose, though not digested, has great im porta n c ei n h u ma n n u tri ti o n . l t i s a maj or constituentol fiber, the non-digestable carbohydrate. The functions of dietary fiber include MUCOPOLYSACCHARIDES made are Mucopolysaccharides heteroglycans unitsof sugarderivatives, namely up of repeating amino sugarsand uronic acids. Theseare more known as glycosaminoglycans commonly (GAG). Acetylatedamino groups,besides sulfate and carboxyl groups are generally present in CAC structure. The presence of sulfate and carboxyl groups contributes to acidity of the mol ecul es, maki ng them aci d mucopo ly, sacchari des. are Some of the mucopolysaccharides found in combination with proteins to forrn mucoproteins or mucoids or proteoglycans (Fig.2.l6l. Mucoproteinsmay contain up to 95o, carbohydrate and 5o/" protein. S-D-Glucose Fig. 2.15 : Structure of cellulose (The repeat:r; -- ' may be several thousands).
  • 29. CARBOHYDRATES Hyaluronic acid 23 Mucopolysaccharides essential are components of tissue structure.The extracellularspacesof ti ssue (parti cul arl yconnecti ve ti ssue-carti l age, skin, blood vessels, tendons)consistof collagen and elastinfibersembeddedin a matrixor ground substance. groundsubstance predominantly The is composedof CAC. The i mportantmucopol ysacchari des uoe i ncl hyal uroni caci d, chondroi ti n4-sul fate, hepari n, dermatansulfateand keratansulfate(Fig.Z.'[1. j 'i ' s: -;-s'/ : - -sS 't -'Fig. 2.16 : Diagrammaticrepresentationof a prateoglycan complex. ,i r :r :, | '.i . :,{,ti i i El 'l H yal uroni caci d i s an i mportantGA C found i n the groundsubstance synovi al ui d of j oi nts of fl and vitreoushumor of eyes. it is also presentas a ground substance n connecti veti ssues, i and forms a gel around the ovum. H yal uroni caci d servesas a l ubri cant and shock absorbanti n j oi nts. BToMEDtCAt/ CLtft|ICALCO$CEpTS rlr Glucose is the most important energy source ol carbohgdrates to the mammals (except ruminants). The bulk of dietary carbohydrote(starch)is dlgestedond as finally obsorbed glucose into the body. Ea Dextrose (glucose in solution in dextrorotatory form) is frequently used in medical practice. Rq'CF Fructose is obundantly found in the semen which is utilized by the sperms for energy. Seueral diseoses are associated with carbohydrate.s e.g., diabetes mellitus, glycogen storage diseoses, galactosemia. trs Accumulation of sorbitol and dulcitol in the fissues moy cause certoin pathological conditions e.g. cotaract, nephropothy. t-s' Inulin, a polymer of is t'ructose, used fo ossessrenal function by meosuringglomerular filtration rate (GFR). ue The non-digestiblecarbohydrate cellulose plays a signilicant role in human nutriticsn. These include decreasing the intestinal absorption ol glucose and cholesterol, qnd increasingbulk of feces to ouoid eonstipation. rt The mucopolysaccharide hyaluronic acid serues a lubricant and shock absorbantin as ioints. The enzgme hgaluronidaseof semen degradesthe gel (contains hyaluronic acid) around the ouum. This qllows eft'ectiue penetration of sperm into the ouum. !3:. [j- IF The mucopolysaccharide heparin is an onticoagulant(preuentsblood clotting). The suruiual of Antarctic lish below -2"C is attributed to the antit'reeze glycoproteins. streptomycin is a glycoside employed in the treatment oJ tuberculosis.
  • 30. B IOC H E MIS TFIY 24 Hy alur o n i c a c i d i s c o mp o s e d o f al ternate unit s of D -g l u c u ro n i c a c i d a n d N-acetyl D- gluc os a mi n e .T h e s e tw o mo l e c u l es form u dis ac c ha ri d e n i ts h e l d to g e th e r b y 0 (t -+ S ) gly c os idi c b o n d (F i 9 ,2 ,1 5 ). H y a l u ro n i c aci d uni c ont ains a b o u t 2 5 0 -2 5 ,0 0 0 d i s a c c h a ri de ts (held by p 1 -+ 4 bonds)with a molecularweight uo t o 4 m i l l i o n . Hyaluronidase is an enzyme that breaks ( B 1 - + 4 l i n k a g e s )h y a l u ro n i c a c i d a n d other CA C. Th i s e n z y m e i s p re s e n t i n hi gh c onc ent r a ti o ni n te s te s ,s e mi n a l fl u i d , and i n certainsnakeand insectvenoms. Hyaluronidase of s em en i s a s s i g n e d a n i m p o rta n t rol e i n f er t iliz at i o n a s th i s e n z y m e c l e a rs the gel ( hy alur on i ca c i d ) a ro u n d th e o v u m a l l ow i ng a better penetration of sperm into the ovum. Hy alur on i d a s e f b a c te ri a h e l p s th e i r i nvasi on o int o t he a n i ma l ti s s u e s . Ghondroitin D-Glucuronicacid N-Acetylglucosamine Hyaluronic acid D-Glucuronic acid N H -C O-C H 3 H N-Acetylgalactosamine 4-sulfate Chondroitin 4-sulfate -o-so3 sulfates Chondroitin 4-sulfate (Greek: chondroscartilage) is a major constituent of various m am m ali a n ti s s u e s (b o n e , c a rti l a g e ,t endons, heart,valves,skin, cornea etc.).Structurally, is it c om par a b l ew i th h y a l u ro n i c a c i d . C h ondroi ti n of units 4-sulfateconsists repeatingdisaccharide c om pos e d o f D -g l u c u ro n i ca c i d a n d N-acetyl 4-sulfate(Fig.2.lV. D-galactosamine oH O-SO; H N-Sulfoglucosamine D-Glucuronate-2-sulfate 6-sulfate Heparin Chondroitin 5-sulfateis also presentin many tissues.As evident from the name, the sulfate group is found on C6 insteadof Ca. t Heparin is composed of alternatingunits of N-sulfoD-glucosamine 6-sulfate and glucuronate 2-sulfate(Fi9.2.17). Dermatan sulfate The name dermatan sulfate is derived from the fact that this compound mostly occurs in the s k in. lt i s s tru c tu ra l l yre l a te d to c h o ndroi ti n -o'r N H _C O_C H . H N-Acetylgalactosamine 4-sulfate sulfate Dermatan Heparin (prevents Heparin is an anticoagulant blood c lot t ing) h a t o c c u rsi n b l o o d , l u n g , l i v e r ,ki dney, t spleen etc. Heparin helps in the releaseof the enz y m e l i p o p ro te i n l i p a s e w h i c h h el ps i n c lear ingt h e tu rb i d i ty o f l i p e m i c p l a s ma. NH-SOa qH2oH o H NH_CO :- N-Acetylglucosamine 6-sulfate Keratansulfate Fiq.2.17 : Structuresof common glycosaminogi',-;-: the disaccharidesas repeating units.
  • 31. 25 Ghapter 2 : CAFIBOHYDHATES Glycosaminoglycan Composition Tissue distribution Function(s) Hyaluronic acid D-Glucuronic acid, Connective synovial tissue, fluid, humor N-acetylglucosaminevitrous Chondroitin sulfate D-Glucuronic bone, blood acid, Cartilage, skin, vessel Helps maintain structure to the N-acetylgalactosamine walls and shapestissues of 4-sulfate lung, kidney, D-Glucuronate 2-sulfate,Blood, liver, spleen Acts ananticoagulant as N-sulfoglucosamine 6-sulfate vessel L-lduronic N-acetyl- Blood valves, valves, Maintains shapes tissues acid, heart the of galactosamine 4-sulfate skin Heparin Dermatan sulfate Keratan sulfate D-Galactose, N-acetyl- Cartilage, cornea, connective glucosamine 6-sulfate tissues 4-sulfate.The only differenceis that there is an inversion in the configuration around C5 of D- glu c u ro n i c a c i d to fo rm L -i d uroni c aci d (Fi9.2.1V. Serves a lubricant. as and shock Promotes absorber. wound healing Keeps cornea transparent and receptors.A selected list of glycoproteins and their major functions is given in Table 2.4. The carbohydratesfound in glycoproteins include mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine,xylose, Keratan sulfate L-fucoseand N-acetylneuraminic acid (NANA). It is a heterogeneous CAG with a variable NANA is an importantsialic acid (SeeFig.2,l1). sulfate content, besides small amounts of Antifreeze glycoproteins : The Antarctic fish mannose, fructose, sialic acid etc. Keratan live below -2oC, a temperatureat which the sulfateessentially consists alternating of units of D-galactosamine and N-acetylglucosamine 6-sulfate. A summary of the glycosaminoglycans with regardto composition,distributionand functions is given in Table 2.3. Several proteins are covalently bound to which are referred to as glycocarbohydrates proteins. The carbohydrate content of glycoproteinvariesfrom 1o/o 90o/o weight, to by Sometimes the term mucoprotein is used for glycoprotein with carbohydrate concentration more than 4"/o. Clycoproteins are very widely distributed in the cells and perform variety of f unc t io n s .T h e s ei n c l u d e th e i r ro l e a s enzymes, hormones,transportproteins,structuralproteins Glycoprotein(s) Collagen proteases, Hydrolases, glycosidases Ceruloplasmin lmmunoglobulins glycoproteins Synovial Thyrotropin, erylhropoietin group Blood substances Fibronectin, laminin Intrinsic factor Fibrinogen Major function(s) Structure Enzymes Transport Defense against infection Lubrication Hormones Antigens Cell-cell recognition and adhesion Absorption ofvitamin 8,, Blood clotting
  • 32. 26 blood would {reeze.lt is now known that ihese fish contain antifreeze glycogtrateinwhich lower the freezingpoint of water and interfere with tne crystalformationof ice. Antifreezegiycoproteins c ons isto f 5 0 re p e a ti n gu n i ts o f th e tri pepti de, alanine-alawine-threonine. Each threonine r es idu e i s b o u n d to B-g a l a c to s yl(1 + 3) o( galactosam N-acetyl i ne. ElIOCHEMISTF|Y ri#i .f*i # CA '? !"r.Ii $: F"r f i { r,.4F. 1.* i4 t: * :il "'.3 The blood group antigens (of erythrocyte membrane) contain carbohydrates as glycoprotei nsor gl ycol i pi ds.N -,A .cetyl gai actosa m ine, gal actose, fucose,si al i c aci d etc. are found in the blood group substances. The carbohydrate content al so pl ays a determi nant e i n bl oo d rol E roup!n8. X. Carbohydrs,tes the polyhydroxyaldehydes ketones,or campounds which produce are or them on hydrolysis. The term sugor is applied to carbohydratessoluble in water and stDeet to taste. Carbahgdratesqre the major dietary energy sources, besides their inualuement in cell structure and uariousother t'unctions. 2. Carbohydrqtesare broadly c/ossiJiedinta 3 groups-ffionasqccharides,oligosoccharides and ytoiysaccharides. The monosacchsrides further diuided into dit't'erent are categories bqsed an the presence af t'wnctional groups {oldosesar ketoses)and the number of carbon atoms (trioses, tetroses,pentases, hexosesand heptcses). 3. Glyceraldehyde{triose) is the simplest carbohydrate and is chosen as a reJerenceto write the cont'iguratian of all other rnonasaccharides (D- anc L- forms). It' two rnonosaccharides differ in their structure around o single carbon atom, they ore known as eplmers.Glucoseand galactose are C4-epimers. 4. D'Glucose is the most predominant naturally occurring aldosdmonosaccharide. Giucoseexisfs cs a and p anemers with dit'Jerentoptical rotations. The interconuersion of a and B anomeric forms with changein the optical rotatian is knoun as mutsratation. 5. Manosaccharides pariicipate in seuercl recctions" These include oxidation, reduction. dehydration, asazone formetion etc. Formatian ol esters and glycosides by manosacchqrides af special significanceln biochemical reactions. is 6. Among the oligosacchqrides, disoccharides are the most common. These include the reducing disaccharides namely lactose(rnilk sugar)and maltase (malt sugar)and the non-reducingsucrose(cane sugar). 7. Palysacclwrides the poiymers ot' monosaccharides their deriuatiues, are or held together by glycosidic bonds. Homopalysaccharides sre compased ot' a single manosaccharicle (e.g., starch, glycogen, cellulose, inulin). Heteropolysaccharides contain a mixture af or Jew monasaceharides thetr derluatiues(e.g., rnucapolysacaharides). 8. Slorch and glgcogensre the carbohydrate reserues plants and animals respectiuelg. ot' Cellulose,exclusiuelyt'ound in plants, is the structural constituent. Inulin is utilized to ossesskidney tunction bg measuring glomerular t'iltration rate (GFR). 9. Mucopoiysaccharides(glycosominoglycans) are the essential companents o/ tlssue structure. They prouide the mstrix or grownd substance extracellular tissue spaces of in whtch collagen and elastin fibers 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 (7-900/o), composition of carbohyd.rate caualently bound to protein. Seueral enzyrnes, hormanes, structura! proteins and cellular receptorsare in fact glycoproteins.
  • 33. Ghapter 2 : CAFIBOHYDHATES I. 27 Essayquestions Add a note on the functionsof with suitableexamples. 1. Define and classifycarbohydrates carbohydrates. and functionsof mucopolysaccharides. the structure 2. Describe to with of configuration monosaccharides, specialreference 3. Cive an accountof the structural glucose. important disaccharides. and functionsof 3 biochemically 4. Discuss structure the of the structure 3 homopolysaccharides. and 5. Define polysaccharides describe II. Short notes (fl (d) derivatives, (c) (b) formation, Clycosidicbond, (e) Sugar (a) Epimers, Mutarotation, Osazone (j) (i) (h) Amino su8ars, Inversion sucrose, Deoxysugars. of (g) Anomers, Enediol, III. F ill i n th e b l a n k s g 1. N a me a n o n -re d u c i nd i s a c c hari de that is taken as a referencefor writing the configurationof others 2. The carbohydrate arounda singlecarbonatom, they are known differ in configuration 3. lf two monosaccharides as are to 4. The s and B cyclic forms of D-glucose referred as is moietyfound in glycosides known as 5. The non-carbohydrate antibiotic 6. Cive an exampleof a glycoside pointsin the structure starchare of bondsat the branching 7. The glycosidic of for employed the assessment kidneyfunction B . The polysaccharide of as that 9. The glycosaminoglycan serves a lubricantand shockabsorbant joints and of 10. Name the sialicacid, mostlyfound in the structure glycoproteins glycolipids IV. Multiple choice questions arounda singlecarbon,namely differ in structure and deoxyribose 11. Ribose (a) Cr (b) Cz (c) C: (d) Cq. 12. O n e o f th e fo l l o w i n gi s n o t a n al dose (c) (a) Clucose(b) Calactose Mannose(d) Fructose. as that 13. The glycosaminoglycan serves an anticoagulant sulfate. sulfate(d) Dermatan (a) Heparin(b) Hyaluronicacid (c) Chondroitin bonds of is 14. The following polysaccharide composed B-glycosidic (a) Starch(b) Clycogen(c) Dextrin (d) Cellulose. formation 15. The carbonatomsinvolvedin the osazone (a) 'l and 2 (b) 2 and 3 (c) 3 and 4 (d) 5 and 6.
  • 34. Lirpirdls The Jat speaks fl ?"'-oR--c-o1H CH2-H i fr : "ffith uater, I say, 'Touch me not': T'otlte tongue,I am tasteful; R3 IY'ithin limits, I am datiful; I fn excess, am dangerous!" (Creek: lipos-fat) are of Breat 1 . Simple lipids : Estersof fatty acids with I ipids L im por t a n c e to th e b o d y a s th e chi ef al cohol s.Theseare mai nl y of tw o types concentrated storage form of energy, besides (a) Fatsand oils (triacylglycerols)Theseare : t heir r ole i n c e l l u l a rs tru c tu re n d v a ri o u sother a of fatty acids with glycerol. The esters bioc hem ica l fu n c ti o n s . As s u c h . l i o i d s are a difference between fat and oil is only heterogeneous group of compounds ano, physical.Thus, oil is a liquid while fat is therefore, it is rather difficult to define them a solid at room temperature. pr ec is elv . Lipids may be regarded as organic substances relatively insoluble in water, soluble in organic solvents (alcohol, ether etc.), actually or potentially related to fatty acids and utilized by the living cells. (b) W axes: E sters fatty aci ds (usual l yl ong of chai n) w i th al cohol sother than gl ycerol . These al cohol s may be al i phati c or al i cycl i c.C etylal cohol i s most commonl y found i n w axes. 2. C ompl ex(or compound)l i pi ds: Theseare , Unlik e th e p o l y s a c c h a ri d e s p ro te ins and estersof fatty aci ds w i th al cohol s contai ni ng nuc leic aci d s , l i p i d s a re n o t p o l y me rs .Further, addi ti onal groups such as phosphate, lipids ar e m o s tl y s ma l l mo l e c u l e s . ni trogenous base, carbohydrate,protei n etc They are furtherdi vi ded as fol l ow s (mo d i fi edfrom Lipids a re b ro a d l y c l a s s i fi e d B loor ) into s i mp l e , c o m p l e x , d e ri v e d and m is c ellan e o uis i d s ,w h i c h a refu rth e r u bdi vi ded l p s into differentgroups 28 (a) P hosphol i pi ds: They contai n phosphor,c base aci d and frequentl ya ni trogenous Thi s i s i n addi ti on to al cohol and fai :. aci ds.
  • 35. Chapter 3 : LIPIDS 29 (i) Glycerophospholipids phospho: These 5. Lipids protectthe internalorgans,serveas lipids contain glycerol as the alcohol insulatingmaterialsand give shape and smooth e .9 .,l e c i th i n , e p h a l i n . c appearance the body. to (ii) Sphingophospholipids : Sphingosine is the alcohol in this group of phosphol i p i d s e .g ., s p h i n g o myel i n. (b) Glycolipids: These lipids contain a fatty Fatty acids are carboxylic acids with acid, carbohydrate and nitrogenous base. hydrocarbonside chain. They are the simplest T h e a l c o h o l i s s p h i n g o s i n e, hence they form of l i pi ds. a re a l s o c a l l e d a s g l y c o sphi ngol i pi ds. Clycerol and phosphateare absent e.g., Occurrence cerebrosides, gangliosides. Fattyacids mainly occur in the esterified form (c) Lipoproteins Macromolecular : complexes as major constituents various lipids. They are of of lipids with proteins. also present as free (unesterified) fatty acids. (d) Other complex lipids: Sulfolipids, amino- Fattyaci ds of ani mal orgi n are much si mpler in l i p i d sa n d l i p o p o l y s a c c h a ri des among are structure in contrast to those of plant origin th e o th e r c o m p l e x l i p i d s . which often contain groupssuch as epoxy, keto, 3. Derived lipids: These are the derivatives hydroxy and cyclopentanerings. obtainedon the hydrolysis group 1 and group of 2lip i d s w h i c h p o s s e s sth e c h a racteri sti cs of Even and odd carbon fatty acids lipid s .T h e s ei n c l u d eg l y c e ro la n d o theral cohol s, Most of the fatty acids that occur in natural fatty acids, mono- and diacylglycerols, lipid (fat) lipids are of even carbons (usually 14C-2OC). soluble vitamins, steroid hormones, hydroThis is due to the fact that biosynthesis fatty of carbons and ketone bodies. acids mainly occurswith the sequential addition 4. M i s c e l l a n e o u sl i p i d s : T h e se i ncl ude a of 2 carbon units. Palmitic acid (l6C) and large number of compounds possessingthe stearic acid (l$C) are the most common. Among characteristics of lipids €.g., carotenoids, the odd chain fatty acids, propionic acid (3C) squalene,hydrocarbons (in and val eri c aci d (5C ) are w el l know n. such as pentacosane bees wax), terpenes etc. N EU T R A T L IPID S: T h e l i p i ds w hi ch are Saturated and unsaturated unchargedare referred as neutrallipids.These fatty acids to are mono-, di-, and triacylglycerols, cholesterol Saturatedfatty acids do not contain double and cholesterylesters. bonds,while unsaturated fatty acids contain one or more double bonds. Both saturated and Functions of lipids unsaturated fatty acids almost equally occur in Lipids perform severalimportant functions the natural lipids. Fatty acids with one double and those with 2 or 1. They are the concentrated fuel reserveof bond are monounsaturated, more double bonds are collectivelv known as the body (triacylglycerols). polyunsaturated fafty acids (PIJFA). 2. Lipids are the constituentsof membrane structure and regulate the membrane Nomenclature of fatty acids (p s per m e a b i l i ty h o s p h o l i p i d a n d c hol esterol ). The naming of a fatty acid (systematic name) 3. They serve as a source of fat soluble is based on the hydrocarbonfrom which it is v it am i n s(4 , D , E a n d K ). derived. The saturatedfatty acids end with a 4. L i p i d sa re i mp o rta n ta s c e l l u l ar metabol i c suffix -anoic (e.g., octanoic acid) while the (steroid regulators hormonesand prostaglandins). unsaturatedfatty acids end with a suffix -enoic
  • 36. 30 BIOCHEMISTF|Y (e.9., octadecanoic acid). In addition to systematic names/ fatty acids have common nameswhich are more widely used (Iable J. l). Length of hydrocarbon cha:n of fatty acids Numbering of carbon atoms : lt starts from the carboxylcarbon which is taken as number 1. The carbonsadjacentto this (carboxylC) are 2, 3, 4 and so on or alternatelya, F, T and so on. The terminal carbon containingmethyl group is known omega (or) carbon. Starting from the methyl end, the carbon atoms in a fatty acid are numberedas omega 1, 2, 3 etc. The numbering of carbon atoms in two different ways is given below 7654 3 2 1 cH3 - cH2 - cH2- cH2-cH2 - cH2 - COOH (t)6 01 a2 o)3 ()4 ol5 Common Name Systematic name Depending on the length of carbon chains, fatty acids are categorized into 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 number of carbon atomsare written first, followed by the nunrber of double bonds and finally the (first carbon) position of Abbreviationx Structure l. Saturated aclds fatty Acetic acid Propionic acid Butyric acid Valeric acid Caproic acid Caprylic acid Capric acid Lauric acid Myristic acid Palmitic acid Stearic acid Arachidic acid Behenic acid Lignoceric acid Ethanoic acid n-Propanoic acid n-Butanoic acid n-Pentanoic acid n-Hexanoic acid n-Octanoic acid n-Decanoic acid n-Dodecanoic acid n-Tetradecanoic acid n-Hexadecanoic acid n-Octadecanoic acid n-Eicosanoic acid n-Docosanoic acid n-Tetracosanoic acid 2:0 3:0 4:0 6:0 8:0 10:0 12:0 14:0 16:0 18: 0 20:0 22:0 24:0 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 16:1;9 18:1;9 18: 2;9, 12 = CH(CHz)zCOOH CHg(CHz)sCH = CH(CHz)zCOOH CHs(CHz)zCH = CHCHzCHCH(CHz)zCOOH = CHg(CHz)+CH F .n ll. Unsaturated acids fatty Palmitoleic acid Oleic acid Linoleic ** acid Linolenic *x acid Arachidonic acid cr1s9-Hexadecenoic acid cls-9-Octadecenoic acid cls,cls-9,12-Octadecadienoic acid Allce9,12,15-0ctadecatrienoic acid Allcls-5,8,11,14- '15 18: 3;9,12, = CHCHzCH = CHCHzCH CHoCHzCH = CH(CHz)zCO0H = CHCHzCH = CHCHzCH 20:4;5,8,11,14 CHg(CHz)+CH Elc0:a!tr3e!o!1ci1___ __=9H9'tcl=_cl9F!)49oli * Total nunber carbon posrtionthe of atons, followed the by number double bonds thefirct and ot carbon ot double bond(s). ** Essential acids. faw
  • 37. 31 Ghapten 3 : LIPIDS double bonds, startingfrom the carboxyl end. Thus,saturated fatty acid, palmitic acid is written as . l 6: 0 , o l e i c a c i d a s 1 8 :1 ;9 , a rachi doni c ac id as 2 0 : 4 ; 5 , 8 , 1 1 , 1 4 . There are other conventionsof representing th t he dou b l e b o n d s .Ae i n d i c a te s a t the doubl e bond is between 9 and 10 of the fatty acid. o 9 representsthe double bond position (9 and 10) from the <oend. Naturallyoccurring unsaturated fatty acids belong to ro 9, ol 6 and o 3 series. a 3 s e ri e s L i n o l e n i c c i d (18 : 3 ;9 , 12, 15) a a 6 series Linoleic acid ('l8 : 2; 9, 12) and a ra c h i d o n i c a c i d (2 0 : 4; 5, 8, 11, 14) r o9 s e ri e s O l e i c a c i d (1 8 : 1 ; 9 ) The biochemically important saturatedand unsaturated fatty acids are given in the Table 3.1. H..ar(CHz)zCOOH H'c'1cHr;rcu, Oleic acid (cls form) Elaldic acid (frans form) Fig. 3.1 : Cis-trans isomerism in unsaturated fattv acids. on the posteriorand lateralpartsof limbs,on the back and buttocks,loss of hair and poor wound heal i ng. lsomerism in unsaturated fatiy aeids Unsaturated fatty acids exhibit geometric isomerismdepending on the orientation of the groups around the double bond axis. lf the atoms or 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 The fatty acids that cannot be synthesized by configuration. Thus oleic acid is a cis isomer the body and, therefore, should be supplied in while elaidic acid is a trans isomer,as depicted fatty acids (EFA). in Fig.3.1 Cis isomers are less stable than frans the diet are known as essential . Chemically, they are polyunsaturated fatty i somers. Most of the natural l y occurri n g acids, namely linoleic acid (18 : 2; 9, 12) and unsaturated fatty acids exist as crs isomers. Iinolenic acid (18 : 3; 9, 12, 15). Arachidonic In the cis isomericform, there is a molecular ac id ( 20 :4 ;5 ,8 , 1 1 ,1 4 ) b e c o m e sessenti ali,f bi ndi ng at the doubl e bond. Thus, ol ei c aci d its precursorlinoleic acid is not provided in the exi sts i n an L-shapew hi l e el ai di c aci d i s a diet in sufficientamounts.The structures EFA of strai ght chai n. Increasen the numberof doubl e i are given in the Table 3.1. bonds w i l l cause more bends (ki nks) and Biochemical basis for essentiality: Linoleic arachi doni c d w i th 4 doubl e bondsw i l l have aci ac id an d l i n o l e n i c a c i d a re e s s e nti al si nce a U-shape. lt is believed that cis isomersof fatty hum an s l a c k th e e n z y me s th a t c a n i ntroduce acids with their characteristic bonds will compactly pack the membranestructure. double bonds beyond carbons 9 to 10. Functions of EFA: Essentialfatty acids are required for the membrane structure and function, transport of cholesterol,formation of lipoproteins,preventionof fatty liver etc. They are also needed for the synthesisof another important group of compounds, namely eicosanoids(Chapter 32. Hydroxy fatty acids: Some of the fatty acids p-Hydroxybutyric are hydroxylated. acid, one of the ketone bodies produced in 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 Deficiency of EFA: The deficiency of EFA structuresare rather rare e.g./ chaulmoogric acid results in phrynoderma or toad skin, found in chaulmoogra oil (used in leprosy characterized the presence horny eruptions treatment)contains cyclopentenylring. by of
  • 38. 32 B IOC H E MIS TFIY o U C H 2 -O -C Fl , A ltl R2-C-O-CH O ttl cH2-o-c-R3 Triacylglycerol o cH2-o-c-R, Rz-C-o-cH I cH2oH 1,2-Diacylglycerol o cH2-o-c -B tHO_CH I cH20H 1-Monoacylglycerol C H ,_OH O ill R -C -O-C H 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 of glycerol, are known (Fi5.3.2). Among these, eicosapolyenoic fatty acids and include prosta- triacylglycerols are the most important glandins ,p ro s ta c y c l i n s e u k o tri e n e a n d throm- bi ochemi cal l y. l, s boxanes. They are discussed together(Chapter 32). Simpletriacylglycerols contain the sametype of fatty acid residueat all the three carbonse.g., tristearoylglycerol or tristearin. Mixed triacylglycerols are more common. (formerly triglycerides) are They contain 2 or 3 different types of fatty acid Triacylglycerols In the esters of glycerol with fatty acids. The fats residues. general,fatty acid attachedto C1 is plants saturated,that attached to C2 is unsaturated and oils that are widely distributedin both and anima l s a re c h e mi c a l l y tri a c y l g l ycerol s. while that on C3 can be either. Triacylglycerols T hey ar e i n s o l u b l e i n w a te r a n d n o n -pol ar i n are named according to placement of acyl 2-l characterand commonly known as neutral fats. radi calon gl ycerole.9.,' l,3-pal mi toyl i nol eoyl gl ycerol . Fats as stored fuel : Triacylglycerols are the m os t abu n d a n t g ro u p o f l i p i d s th a t p ri mari l y Triacylglycerols of plants, in general, have function as fuel reservesof animals. The fat higher content of unsaturated fatty acids reserveof normal humans (men 2Oo/o, women compared to that of animals. 25% by weigh$ is sufficientto meet the body's $tereospecific numbering caloric requirements 2-3 months. for of gl ycerol Fats primarily occur in adipose tissue: Adipocytes of adipose tissue-predominantly The structureof glycerol gives an impression found in the subcutaneouslayer and in the that carbons1 and 3 are identical. This is not true abdominalcavity-are specialized storageof in a 3-dimensional for structure. order to represent In triacylglycerols. The fat is stored in the form of the carbon atomsof glycerol in an unambiguous globulesdispersedin the entire cytoplasm.And manner, biochemists adopt a stereospecific surprisingly, triacylglycerols not the structural numbering(sn) and prefix glycerolwith sn. are componentsof biological membranes. Structures of acylglycerols Monoacyl: glycerols, diacylglycerols and triacylglycerols, respectivelyconsisting of one, two and three moleculesof fatty acids esterified a molecule to 6n,on no-C'.-H 6tr,ot sn-GfcJrol
  • 39. C*rapter'3 : LIPIDS 33 It should be noted that C1 and C3 are a. tipid peroxidation in vivo: In the living d ifferent. Cells possess enzymes that can cel l s, l i pi ds undergo oxi dati on to produce dis t ingui s h th e s e tw o c a rb o n s. Thus peroxidesand free radicalswhich can damage glycerokinase phosphorylates sn-3 (and not sn-l) the tissue.The free radicalsare believedto cause glycerol to give sn-glycerol3-phosphate. inflammatory diseases, ageing, cancer/ atherosclerosis etc. lt is fortunatethat the cells PROPERTIES TRIACYLGTYCEROLS possess OF antioxidants such as vitamin E, urateand superoxide dismutaseto prevent in vivo lipid A few importantproperties triacylglycerols, of peroxidation (Chapter 34). which have biochemical relevance, are discussed below 1. Hyd ro l y s i s T ri a c y l g l y c e ro l s undergo : stepwiseenzymatic hydrolysisto finally liberate free fatty acids and glycerol. The process of hydrolysis,catalysedby lipasesis importantfor digestionof fat in the gastrointestinal tract and fat mobilization from the adiposetissues. Tests to check purity of fats and oils Adulterationof fats and oils is increasing day by day. Several tests are employed in the laboratoryto check the purity of fats and oils. Some of them are discussed hereunder lodine number : lt is defined as the grams 2. Saponification The hydrolysisof triacyl: (number) of iodine absorbedby 100 g of fat or glycerolsby alkali to produceglyceroland soaps oil. lodine number is usefulto know the relative is k nown a s s a o o n i fi c a ti o n . unsaturation fats, and is directly proportional of Triacylglycerol+ 3 NaOH ---------+ to the content of unsaturated fatty acids. Thus Clycerol + 3 R-COONa(soaps) lower is the iodine number, lessis the degreeof The i odi ne numbersof common 3. Rancidity: Rancidity is the term used to unsaturati on. represent the deterioration of fats and oils oils/fatsare given below. resultingin an unpleasant taste. Fatscontaining FaUoil lodine number unsaturated fatty acids are more susceptible to r anc idit v . Coconut oil 7- 10 Butter 25- 28 Rancidity occurs when fats and oils are Palm oil 4C 55 exposed to air, moisture, light, bacteria etc. Olive oil 80- 85 Hydrolytic rancidity occurs due to partial Groundnut oil 85- 100 hydrolysis of triacylglycerols by bacterial Cottonseed oil 100 110 enzymes.Oxidative rancidity is due to oxidation Sunflower oil 125 135 of unsaturatedfatty acids. This results in the Linseed oil 175-200 formation of unpleasant products such as dicarboxylic acids, aldehydes, ketones etc. D etermi nati on i odi ne number w i l l hel p to of Rancid fats and oils are unsuitablefor human know the degree of adulterationof a given oil. c ons um o ti o n . Saponificationnumber : lt is defined as the Antioxidants : The substanceswhich can mg (number) of KOH required to hydrolyse preventthe occurrenceof oxidativerancidityare (saponify) one gram of fat or oiL Saponification known as antioxidants. Trace amounts of number is a measureof the averagemolecular ant iox ida n tss u c h a s to c o p h e ro l s(v i tami n E ), size of the fatty acids present. The value is higher hy dr oqu i n o n e ,g a l l i c a c i d a n d c ,-n a p hthol are for fats containing short chain fatty acids. The addedto the commercialpreparations fats and saponification of numbersof a few fats and oils are oils to preventrancidity.Propylgallate,butylated given below hydroxyanisole(BHA) and butylated hydroxyH umanfat : 195-200 toluene (BHT) are the antioxidantsused in food Butter :230-240 preservation. Coconut oil : 250-260
  • 40. 34 E l IOC H E MIS TR Y (RM) number: lt is definedas Reichert-Meissl the number of ml 0.1 N KOH required to completely neutralize the soluble volatile fatty acids distilledfrom 5 g fat. RM number is useful in testingthe purity of butter since it containsa good concentration volatilefatty acids (butyric of ac id, c apr oic a c i d a n d c a p ry l i c a c i d ).T h i s i s i n contrast to other fats and oils which have a negligibleamount of volatile fatty acids. Butter has a RM nu m b e r i n th e ra n g e2 5 -3 0 ,w h i l e i t i s les st han I f o r mo s t o th e r e d i b l e o i l s . T h u s any adulteration of hutter can be easily tested by t his s ens it iv e M n u m b e r. R Acid number : lt is defined as the number of mg of KOH required to completely neutralize free fatty acids presentin one gram fat or oil. In normal circumstances, refinedoils should be free from any free fatty acids. Oils, on dec om oos it i o n -d u e to c h e mi c a l o r b a cteri al contamination-yield free fatty acids.Therefore, oils with increasedacid number are unsafefor hum an c ons u m p ti o n . There are two classes phospholipids of (or phosphogl yce1. C l ycerophosphol i pi ds rides)that contain glycerol as the alcohol. (or 2. S phi ngophosphol i pi ds sphi ngomyel i ns) that contai n sphi ngosi ne the al cohol . as 1. i t " .t .;:i r ,. : . ,,,.i ., i - l , are the maj or l i pi ds C l ycerophosphol i pi ds membranes. They consist that occur in biological glycerol 3-phosphate at its C1 and esterified of C 2 w i th fatty aci ds. U sual l y, C 1 contai ns a saturated fatty acid while C2 contains an fatty acid. unsaturated 1 . Phosphatidic acid : This is the simplest phosphol i pi d. l t does not occur i n good concentration in the tissues. Basically, phosphati di c aci d i s an i ntermedi atei n the s synthesi s tri acyl gl yceroland phosphol i pi ds. of contai ni ng The other gl ycerophosphol i pi ds basesor other groups may differentnitrogenous be regardedas the derivativesof phosphatidic aci d. These are 2. Lecithins (phosphatidylcholine)z groupof phosphol i pi dsn the i the mostabundant cel l membranes. hemi cal l y,l eci thi n (C reek : C These are complex or compound lipids lecithos-egg yolk) is a phosphatidicacid with phosphoric containing acid,in addition fatty chol i ne as the base. P hosphati dyl chol i nes to acids, nitrogenous base and alcohol (Fig.3.3). represent the storage form of hody's choline. BtoMEDtCAL/ CLtNtCAt CONCEpTS os Lipids are important to the body as constituentsof membranes,source ol fat soluble (A, D, E and K) uitamins qnd metabolic regulators (steroid hormones and prostaglandlns), e Triacylglycerols (fots) primarily stored in the adipose tissue ore concentrated t'uel reseruesof the body. Fats t'ound in the subcutoneous tissue and around certaln orgons serue os thermal insulators, se The unsaturated fatty acids-linoleic and linolenic acid-<re essentiolto humans, the deficiency of which cousesphrynodermo or toad skin. s The cyclic fatty acid, namely choulmoogricocid,is employed in the treatment of leprosy. og Fqts and oils on exposureto ah; moisture, bacteria etc. 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--are commonly used. *
  • 41. 35 Chapter 3 : LIPIDS o ll g ,11 ill cH2-o-c-R1 ill .:1 RI-C-O-CH -l CH2- i- ' - r ' - i' t (1) Phosphatldicacid (phosphatidylcholine) tz) Leclthln i o tl I C H 2 -O -C -R 1 ill rf R2-C-O-qH CH2-C--l-CH2-CH2-NH2 Ethanolamine C(3) Cephalln (phosphatidylethanolamine) ,E myalnositol (4) Phosphatidyllnosltol o tl cH2-o-c-Rl ? R2-c-o-?H {l A l tl ,t_ _ C- .),f,l (5)Phosphatldylserlne l- CH2-.i Ethanolamine (6) Plasmalogen(phosphatidalelhanolamine) r , n- cH2 ? tr. Hc-o-c-R3 ? cH2-o-c-R1 cH2-, ? I H?-OH R2-C-O-CH ^ .:1 -CHz-CH2-NH2 CH2-t', - i' i--- CH2-r-:- = C-CHz-CH-COO- o QH2-O-CF{=CH-Rl R2-C-O-CH ,: r-.'-CHe + R4-C-O-CH2 I ehospnatioytgty."ro, (diphosphatidylglycerol) (7)Cardlollpin lCeramid" _ (/t'soninoosrne$)> CH3-(CH2)12-CH:CH-CH-?H-NH-C-R ' , *.CHg -CHz-CHz-Nf9,Tt r_ (8) Sphlngomyelln Choline of Fig. 3.3 : Sttuctures phospholipids. 'n3
  • 42. 36 BIOCHEMISTF|Y (a) Dipalmitoyl lecithin is an important ph o s p h a ti d y l c h o l i n e u n d i n l u n gs,l t i s a fo surface active agent and prevents the adherence of inner surface of the lungs due to surfacetension. Respiratory distresssyndrome in infants is a disorder characterized the absence dipalmitoyl by of lec i th i n . by an amide linkage to a fatty acid to produce ceramide.The alcohol group of sphingosineis i bound to phosphoryl chol i nen sphi ngomyel i n (Fig.3.3). Sphingomyelins important are structure of constituents myelin and are found in good quantity in brain and nervoustissues. (b) Lysolecithinis formed by removal of the fatty acid either at C, or C, of lecithin. are Phospholipases a group of enzymesthat There are four distinct hydrolysephospholipids. (A phosphol i pases r, 42, C and D ), each one of them specificallyacts on a particularbond. For details, refer lipid metabolism(Chapter l4). 3. Cephafins (phosphatidylethanolamine) : Ethanolamine the nitrogenous is base presentin c ephalin sT h u s ,l e c i th i na n d c e p h a l i nd i fferw i th , regard to the base. Action of phospholipases Functi ons of phosphol i pi ds 4. Phosphatidylinositol The steroisomer : P hosphol i pi ds consti tutean i mportantgroup myo-inositolis attachedto phosphatidicacid to of compound lipids that perform a wide variety giv e phos p h a ti d y l i n o s i to l (P lh i s i s a n i mportant of functions T ). me mb ra n e s . h e a cti on of T c om Done n to f c e l l phosphol i pi ds w 1. In associ ati on i th protei ns, (e.9. oxytocin, vasopressin) certain hormones is form the structural components of membranes m ediat edth ro u g h Pl . and regulatemembrane permeability. 5. P ho s p h a ti d y l s e ri n e : T h e a mi n o aci d 2. P hosphol i pi ds (l eci thi n, cephal i n and s er ine is p re s e n ti n th i s g ro u p o f g l y cerophos- cardi ol i pi n)i n the mi tochondri a are responsi bl e pholipids. Phosphatidylthreoninealso found in for maintaining the conformation of electron is certaintissues. transportchai n components,and thus cel l ul ar 6. Plasmalogens When a fatty acid is respiration. : attachedby an ether linkageat C1 of glycerol in participate the absorption in 3. Phospholipids gly c e ro p h o s p h o l i p i d s , th e t he resul tant of fat from the intestine. c om poun d i s p l a s m a l o g e n . P h o s phati dal 4. P hosphol i pi ds are essenti al for the et hanola mi n ei s th e m o s t i mo o rta n t w hi ch i s synthesi sof di fferent l i poprotei ns,and thus similar in structureto phosphatidylethanolamine participate in the transport of lipids. but for the ether linkage (in place of ester).An 5. Accumulationof fat in liver (fattyliver)can unsaturatedfatty acid occurs at C1. Choline, inositol and serine may substitute ethanolamine be preventedby phospholipids,hence they are regarded as lipotropic factors. t o giv e ot h e r p l a s ma l o g e n s . Z. Cardiolipin : lt is so named as it was first isolated from heart muscle. Structurally, a c ar diolip i n c o n s i s ts o f tw o mo l e c ul es of phos pha ti d i c c i d h e l d b y a n a d d i ti o n a lgl ycerol a through phosphate groups. lt is an important c om pone n t o f i n n e r mi to c h o n d ri a lm e mbrane. Car diolip i n i s th e o n l y p h o s p h o g l y ceri de that possesses antigenic properties. fatty acid 6. Arachidonicacid, an unsaturated liberated from phospholipids, serves as a (prostaprecursor the synthesis eicosanoids for of glandins,prostacyclins, etc.). thromboxanes parti ci patei n the reverse 7. P hosphol i pi ds chol esterol transport and thus hel p i n the removal of cholesterolfrom the body. 8. Phospholipidsact as surfactants(agenL. l ow eri ng surface tensi on). For i nstance S phingomy e l i n s is di pal mi toyl phosphati dyl chol i ne an i mportar: Sphingosine an amino alcohol present in fung surfactant. Respiratory distresssyndrome ^ is with insufficientproductio^ s phingomy e l i n(s p h i n g o p h o s p h o l i p i ds). do infantsis associated s They not containglycerolat all. Sphingosine attached of this surfactant. is
  • 43. 37 Chapter 3 r LIPIDS Sphingosine loHlo YIY] o-cH2 Fig. 3.4 : Structure ot galactosylceramide (R = H). Fot sulfagalactosylceramideR is a sulfatide (R = SOi-). 9. Cephalins, importantgroup of phosphoan lipids pa rti c i p a te n b l o o d c l o tti n g . i Ceramide are 10. P h o s p h o l i p i d s (p h o s p h a ti d y l i nosi tol ) membranes. inv olv ed n s i g n a tra n s mi s s i oa c ro s s i l n Glucose I Galactos f tl N-AcetylN-Acetylgalactosamine neuraminic acid C lyco Ii p ids (glycosphingol ipids) are i m portant c ons t it u e n tso f c e l l me mb ra n e a n d nervous are tissues(particularlythe brain). Cerebrosides . t he s im p l e s t rm o f g l y c o l i p i d s T h e y contai n a fo Li poprotei ns are mol ecul ar compl exes of attachgdto a fatty acid) ceramide (sphingosine lipids with proteins. They are the transport and one or more sugars. Galactocerebroside vehi cl esfor l i pi ds i n the ci rcul ati on.There are (galactosylceramide) are and glucocerebroside five types of lipoproteins, namely chylomicrons, the most importantglycolipids.The structureof very low density lipoproteins (VLDL), low galactosylceramide given in Fig3.a. lt contains is (LDL), high density lipoproteins density cerebronic acid. the fatty acid Iipoproteins (HDL) and free fatty acid-albumin is Sulfagalactosylceramide the sulfatide derived complexes. Their structure, separation, from galactosylceramide. metabolismand diseases are discussed together found (Chapter l4). Gangliosides Theseare predominantly : in ganglionsand are the most complex form of glycosphingolipids. They are the derivativesof and contain one or more molecules cerebrosides of N- ac e ty l n e u ra m i n i a c i d (N A N A ), the most c o im oor t a n ts i a l i c a c i d . T h e s tru c tu re f N A N A i s Steroids are the compounds containing a given in carbohydrate chemistry(ReferFig.2.l1. cycl i c steroi d nucl eus (or ri ng) namel y The most important gangliosidespresent in cyclopentanoperhydrophenanthrene (CPPP). lr t he br a i n a re C M1 , C M2 , C D , a nd C T, consistsof a phenanthrenenucleus (rings A, B (G represents ganglioside while M, D and T and C ) to w hi ch a cycl opentaneri ng (D ) i s indicate rnono-, di- or tri- sialic acid residues, attached. The structure and numbering of CPPP are and the number denotes the carbohydrate to the ceramide). The shown in Fi9.3.5.The steroid nucleusrepresents sequence attached ganglioside, in CM2 that accumulates Tay-Sachs saturatedcarbons, unlessspecificallyshown as is reoresented next (outline structure). doubl e bonds. The methyl si de chai ns (19 and disease
  • 44. 38 BIOCHEMISTF|Y Structure and occurrence The structure of cholesterol (C27Ha6O)is depicted in Fig.3.5.lt has one hydroxyl group at C3 and a double bond between C5 and C6. An 8 carbon aliphatic side chain is attachedto C17. Cholesterol contains a total of 5 methyl Sroups. Due to the presence of an -OH group, A i chol esterols w eakl y amphi phi l i c. s a structural component of plasma membranes,cholesterol is an important determinant of membrane permeabilityr, properties. The occurrence of of cholesterolis much higher in the membranes I les. sub-celu lar organel with fany Cholesterolis founi in association acids to- form cholestervl esters (esterification occurs at the OH group of C3). Properties and reactions : Cholesterol is an yel l ow i sh crystal l i nesol i d. The crystal s,under the microscope, show a notched (E) appearance.Cholesterol is insoluble in water and sol ubl e i n organi c sol vents such as chloroform, benzene,ether etc. Fig. 3.5 : Sttucturcs of steroids (A, B, C-Perhydroph enanthrene; D-Cyclopentane). Several reactions given by cholesterol are useful for its qualitative identification and quantitative TheseincludeSalkowski's estimation. reaction and Zak's test, Liebermann-Burchard test. 18) attachedto carbons10 and 13 are shown as s ingle bo n d s . At c a rb o n 1 7 , s te ro i d s usual l y Functions of cholesterol: Cholesterol is a c ont ain a s i d e c h a i n . poor conductor of heat and electricity,since it T her e a re s e v e ra ls te ro i d si n th e b i ol ogi cal has a high dielectric constant. lt is present in s y s t em .Th e s e i n c l u d e c h o l e s te ro l ,b i l e aci ds, abundance in nervous tissues.lt appears that cholesterolfunctions as an insulatingcover for vitamin D, sex hormones, adrenocortical of hor m one s ,s i to s te ro l s , a rd i a c g l y c o si desand the transmi ssi on el ectri cal i mpul ses i n the c alk aloid s . l f th e s te ro i d ,c o n ta i n s n e or more nervous tissue. Cholesterol performs several o hy dr ox yl g ro u p s i t i s c o m m o n l y k now n as other bi ochemi calfuncti onsw hi ch i ncl ude i ts role in membranestructureand function, in the s t er ol ( m e a n s s o l i d a l c o h o l ). synthesis of bile acids, hormones (sex and CI{OLESTEROL cortical) and vitamin D (for details, Refer Chofesterol, exclusively found in animals, is Chapters 7 and l9). t he m os t a b u n d a n t a n i ma l s te ro l . l t i s w i del y distributedin all cells and is a major component ERGOSTEROL Ergosterol occurs in plants.lt is also found as of cell membranes and lipoproteins. Cholesterol (Creek: chole-bile) was first isolatedfrom bile. a structuralconstituentof membranesin yeast Cholesterolliterally means 'solid alcohol from and fungi. Ergosterol(Fig.3.5) is an important precursorfor vitamin D. When exposedto light, bile. '
  • 45. fTi li Cfrraptee3 : LIPIDS 39 I the ring B of ergosterol opens and it is converted to ergocalciferol, a compound containing vitamin D activity. cH3(cHdn-cooHydrophiic Hydrophobic hydrocarbonchain carboxyl group (tail) (head) (A) Fatty acid The other sterolspresentin plant cells include stigmasterol and ftsitosterol. o tl Hydrophobic tail A s per defi ni ti on,l i pi ds are i nsol ubl e(hydr ophobi c) i n w ater. Thi s i s pri mari l y due to t he predominant presenceof hydrocarbon groups. However, some of the lipids possesspolar or hydrophi l i cgroupsw hi ch tend to be sol ubl e in water. Molecules which contain both hydrophobicand hydrophilic groups are known as amphipathic (Creek : amphi-both, pathospassi on). HYdroPhilic head (B) Phospholipid Examples amphipathic lipids: Among the of l i pi ds, fatty aci ds, phosphol i pi ds, sphi ngol i pids, bile salts and cholesterol(to some extent) are amphi pathi ci n nature. (C) Amphipathic lipid Aqueous pnase Fattyacids contain a 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. (D)Micelle OOOOC ttttl ltttl Nonpolarphase oo phase Aqueous (E)Lipid bilayer Phospholipids have a hydrophilichead (phosphate group attachedto choline, ethanolamine, inositol etc.) and a long hydrophobic tail. The generalstructure an amphipathicIipid may be of represented a polar or hydrophilic head with as a non-polar or hydrophobictail (Fig.3.6). Orientation amphipathic lipids: When the amphipathic lipids are mixed in water (aqueous Aqueousphase phase), the polar groups (heads) orient themsel ves tow ards aqueous phase w hi l e t he non-polar (tails) orient towards the opposite directions.This leadsto the formation of mr'celles (Fi9.3.6).Micelle formation, facilitated by bile salts is very important for lipid digestion and absorption (Chapter 8). Me*nhrane Fig. 3.6 : Summary of amphipathic lipids in the formation of micelle and lipid bilayer. bilayers In case of biological membranes, bilayer of a l i pi ds i s formed ori enti ngthe pol ar headsto t he
  • 46. B IOC H E MIS TR Y 40 COilCEPTS BIOMEDICAL CLIITIICAL / The phospholipid4ipalmitoyl lecithin-preuents the adherence of inner surface of the lungs, the absence of which is ossociofed with respiratory disfress syndrome in infants. !ei- Cepholinsparticipate in blood clotting. € The action of certain hormones is mediated through phosphatidylinositol. g Phospholipids are important for the synthesis and transport of lipoproteins ond reuerse tronsport ol cholesterol. Cholesterol is essential for the synfhesisol bile ocids, hormones(sexand cortical)and uitamin D. Lipoproteins occur in the membrone structure, besidesseruingos o meansol transport uehiclesfor lipids. ond atherosclerosis. Lipids are associated with certain disorders----obesity out er a q u e o u s p h a s e o n e i th e r s i de and the nonpolar tails into the interior (Fig.3.6. fhe f or m a ti o n o f a l i p i d b i l a y e r i s th e basi s of membranestructure. ar combi nati on w i th ti ssue speci fi c anti B ens, e as carriersof drugs to tarBettissues. used Emulsions These are produced when non: pol ar l i pi ds (e.g. tri acyl gl ycerol s) are mi xed Liposomes They are produced when amphi- with water. The particlesare larger in size and : pat hic l i p i d s i n a q u e o u sm e d i u m a re subj ected stabi l i zed by emul si fyi ng agents (usuallr to sonification. They have intermittent aqueous amphi pathi c l i pi ds), such as bi l e sal ts and phas e s i n th e l i p i d b i l a y e r. L i posomes, i n phosphol i pi ds.
  • 47. LIPIDS substances relatiuely inso,luble in water, soluble in organic actually or potentially related to totti-o"iJl'ord or" utilized 2 Lipids are crassifiedinto simpre (fats c,omprex (phosphoripids, deriued (fatty acids, steriod n"r_"n"rl ,and.,oirs), grgcolipids), and miscelloneous (carotenoids). 3' Fatty acids are the maior constituents of.uarious ripids. saturatedand unsaturoted acids armost equarsoccurin fatty ri'['i":ur"rrirrated ii[i:"0"i:':;::i:i "tt"*i'1io11i, (pr'FA) fatty acids and tinoteni, o,id o," the.s'se,'tiai ioitv o,ia, that needto be 4' Triacylglycerols (simply fats)are the esters glycerol with fatty acids. in adipose tissueand pr:imoriry ,*nrtior.o, 7f They are found ',u"li.r"rr"- oj oli^otr. seuerar tests number, number)are (iodine RM emproged in tn" ioiorrJo"/iio"i"i'r"ine purity of and oirs, fots 5' Phosphotipidsare complex lipids cctntainin.g phosphoric acid. Glycerophospholipids ;;:I:,:Ji:'::;',:i;,:i:;,:ot andthese inct-ude'r"'"it"' i-"intin, phosphatravrinoiitot, 6' sphingophospholipids(sp.hingornyelins) contain sphingosine the as alcohol in place of tJi:;fi,!:,ttvcerophosphoti;idsi.ih-osphoripid;";,;; i;;';";:, constituents ptasma of 7 cerebrosides are the .simprest form of grycoripids which occur in the membranes neruous tissue. Gangriosides of are predominontrg round in ti" t gorstions. Theycontain one or more moteculesof N-acetylneuraminic.";,; ifi;:;r;:,)i." 8. Steroids contain th,ering cyclopentanoperhydrophenanthrene. T importance incrude. r,rr-iir'n," ;;;.'I::#r::t i!^i,"Jlfr,,iZ,, hormones-A steroid "nlutt"ii,""i,,tJ'ir,ar, containing'or."or"*or. hydroxyr groupsis known as steror. 9' cholesterar is the most abundqnt animarsterar.It contains one hydroxyr a double bond (c51) and an group (at cz), co,rao.n side choin,ti"rn.a b cp. "igl,t cholesterol-is ,,,",riiJ"i i,'"ioJ",,,,nesis acids, 2."?;:::::'t:.":"T"#[::; o/ bire :::;,:x:_i:ao,, r0 The lipids that oor:::r both hvdrophobic (non -porar)and hydrophiric (porar) groups known as amphipathic. rhese are in,i"a" iori, o,,;;.;:;;;h;i;,:;:, sphinsoripids and bite ripids on i^poioi constituentsin the biiaeers ';:;r:#::.''athic of the bioroqical
  • 48. 42 B IOC H E MIS TR Y I . Essayquestions 1. Writean account classification lipidswith suitable of of examples. 2. Describe structure the and functionsof phospholipids. 3. Discuss saturated unsaturated acidsof biologicalimportance, the and alongwith theirstructures. fatty 4. Describe structure steroids. the of Add a noteon the functionsof cholesterol. 5. Discuss biological the importance amphipathic of lipids. II. Short notes (a) Structure triacylglycerols, Clycolipids,(c) Essential (b) of fatty acids,(d) Cis-trans isomerism, (e) Rancidity, lodine number,(g) Phosphatidylinositol, Sphingomyelins, Steroidnucleus, (0 (h) (i) (j) Micelles. III. Fill in the blanks 1. The lipidsthatfunctionasfuel reserve animals in 2. The isomerism associated with unsaturated fatWacids 3. The cyclic fattyacid employedin the treatment leprosy of 4. The lipidsthat are not the structural components biologicalmembranes of 5. The prefixsn usedto represent glycerol,sn stands for 6. The number mg of KOH required hydrolyse g fat or oil is knownas of 1 to The phospholipid that prevents adherence innersurfaces lungs the of of B . The phospholipid produces that second messengers hormonal in action 9. Namethe glycolipids containing N-acetylneuraminic acid 10 . The steroids containa cyclic ring knownas IV. Multiple choice questions 11. The nitrogenous basepresent lecithin in (a)Choline(b) Ethanolamine Inositol Serine. (c) (d) 12. The number doublebondspresent arachidonic of in acid (a)1 (b) 2 (c) 3 @)a. 13. On e o f th e fo l l o w i n g s a n a mphi pathilcpi d i i (a)Phospholipids Fatty (b) (d) acid (c) Bilesalts All of the above. 14. Esterification cholesterol of occursat carbonposition (a)1 (b)2 (c)3 (d)4. 15. Namethe testemployedto checkthe purity of butterthroughthe estimation volatilefattyacids of (a)lodinenumber(b) Reichert-Meissl number(d)Acid number. number(c)Saponification
  • 49. rl trii, p rotuint are the most ahundant organic I molecules of the living system. They occur in every part of the cell and constitute about 50'h of the cellular dry weight. Proteinsform the fundamentalbasis of 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)suggested name the proteinsto the group of organic compoundsthat are utmost important to life. Mulder (Dutch chemist)in 1838 used the term proteins for the high mo l e c u l a r w e i g h t n i tro g e n -ri ch and most abun d a n t s u b s ta n c e s re s e n t i n a ni mal s and p olants . Structuralfunctions: Certainproteinsperform brick and mortar roles and are primarily responsible for structureand strengthof body. Theseinclude collagen and elastinfound in bone matrix, vascular system and 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,storage proteins, besides thei r functi on i n geneti c control , muscle contraction,respirationetc. Proteinsperforming dynamic functionsare appropriately regardedas the working horsesof cell. Elermental cornposition clf Broteins Proteins predominantly are constituted five bv major elementsin the following proportion. Functions of proteins Carbon 50 - 55% Proteins performa greatvariety of specialized Hydrogen 6 - 7.3% and e s s e n ti afu n c ti o n si n th e l i v i n g cel l s.These l Oxygen 19 - 24% functions may be broadly grouped as static Nitrogen 13 - 19% (structural) and dynamic. S ul fur 0 - 4o/" 43
  • 50. 44 BIOCHEMISTFIY Besidesthe above, proteins may also contain other elements such as P, Fe,Cu, l, Mg, Mn, Zn etc. The a-carbon atom binds to a side chain by represented R which is differentfor each of The ami no the 20 ami no aci dsfound i n protei ns. The content of nitrogen, an essential acids mostly exist in the ionized form in the component of proteins,on an averageis l6%. biological system(shown above). Estimation nitrogen in the laboratory(mostly of by Kjeldahl's method is also used to find out the Optical isomers of amino acids amount of protein in biologicalfluids and foods. lf a carbon atom is attachedto four different groups, it is asymmetricand thereforeexhibits Proteins are polymers of amano acids opti cal i someri sm.The ami no aci ds (except (with concen- glycine) possessfour distinct groups (R, H, Proteins completehydrolysis on trated HCI for several hours) yield L-cr-amino COO-, NH;) held by c,-carbon.Thus all the ac ids . T h i s i s a c o m m o n p ro p e rty of al l the ami no aci ds (exceptgl yci ne w here R = H ) have proteins.Therefore, proteins are the polymers of optical isomers. Lq"-amino acids. The structures L- and D-amino acids are of written based on the configuration of L- and STANDARD AMINO ACIDS D-glyceraldehyde as shown in Fig.4.l. The As many as 300 amino acidsoccur in natureproteinsare composedof L-c-amino acids. Of these, only 20-known as standard amino of amino acids acids are repeatedly found in the structure of Glassification proteins, isolated from different forms of lifeThere are different ways of classifyingthe anim al, p l a n t a n d m i c ro b i a l .T h i s i s b e cause of amino acids basedon the structure and chemicat the universalnatureof the geneticcode available nature,nutritional fateetc. metabolic requirement, for the incorporation of only 20 amino acids A. Amino acid classification based on the when the proteinsare synthesized the cells. in structure : A comprehensiveclassificationof The processin turn is controlled by DNA, the ami no aci ds i s based on thei r structureand geneticmaterialof the cell. After the synthesis of a chemi calnature.E achami no aci d i s assi gned proteins,some of the incorporatedamino acids 3 letter or 1 letter symbol. These symbols are undergo modifications form their derivatives. to the commonl y usedto represent ami no aci ds i n protein structure.The 20 amino acids found in proteinsare divided into seven distinct groups. ln Table 4.1, the different groups of amino A m ino a c i d s a re a g ro u p o f organi c acids,their symbolsand structures given.The are compounds containing two functional groupsgroups are described salientfeaturesof different amino and carboxyl. The amino group (-NH2) next is basic while the carboxyl group (-COOH) is ac idic in n a tu re . cHo cHo I I General structure of amino acids H -C -OH oH-c-H I I The amino acidsare termedas cr-amino acids, cH2oH cH2oH if both the carboxyl and amino groups are D-Glyceraldehyde L-Glyceraldehyde attachedto the same carbon atom, as depicted R R I below H 2N -C -H H -C -N H 2 I I H H cooH cooH I I L-Amino acid D-Amino acid R-C -C O OH R-C-COOI Fig.4.l : D- and L-forms of amino acid based on the NHz NHJ structure ot glyceraldehyde. Existsas ion General structure
  • 51. Chapter 4 : PFIOTEINS AND AMINO ACIDS 45 Symbol 3 letters I letter Structure Special group present l. Amino aclds withaliphatic chalns side 1. Glycine Gly 2. Alanine Ala 3. Valine Val 4. Leucine Leu H-CH-COOt+ NHi Branched chain t'!r-.rz-QH-coo HgC Branched chain *nl Branched chain 5. lsoleucine (-OH) groups ll. Amino acidscontainlng hydroxyl 6. Serine Ser 7. Threonine Thr cH2-cH-coooH NHi Hydroryl H3C-CH-CH-COO- Hydroryl on rrFrt Tyrosine Tyr See under aromatic Hydroxyl Trble 4.1 contd. nerl page
  • 52. 46 BIOCHEMISTF|Y Symbol Narne 3 letters Special group present Structure I letter lll. Sulfur containing amino acids 8. Cysteine Cys C cH2-cH-cooSH NHi Sulfhydryl cH2-cH-coo- A rrt I Cystine Disulfide I cH2-cH-@ol+ NHi 9. Methionine Met M cH2-cH2-cH-@o- Thioether b-cH. runt lV. Acidic amino acids theiramides and 10.Aspartic Asp acid 11.AsparagineAsn po -ooc-cH2-cH- @o H2N-C-CH2-CH-COO- 6 12.Glutamic Glu acid p-Carboryl r+ NHi Amide nnl Y P ct -ooc-cH2-cH2-?H-coo- yCarboryl NH; 13.Glutamine Gln H2N-C CHz-CH2?H-COO- o Amide NHI V, Basic amino acids 14. Lysine Lys q e6Y P CH2-CH2-CH>-CHt -CH-@Ol+ l; NHi NHi e-Amino NH- CH2- CH2 CH2- CH-@O- 15. Arginine Arg ?:*t; NHt Guanidino NHz 16. Histidine His IINHi lmidazole HNN -rq-cH+-coot.blo 4.1 contd. nort pag€
  • 53. 47 AND AMINO ACIDS ehaptee r* r PFIOTEINS 3 letters Specialgroup present Structure Symbol Name 1 letter acids amino Vl. Aromatic Benzenephenyl or cH2-9H-COO- Phe 17. Phenylalanine Nxt 18.Tyrosine /- Tyr 4-'ir----------c 19. Tryptophan Trp Phenol '*:/""-[Xt"o" W u_/ ''l*r H cH coo2- lndole H Vll. lminoacid 20. Proline Pynolidine Pro gH I (Note B group shown red) in is : 1. Amino acids with aliphatic side chains : These are monoamino monocarboxylic acids. This group consists of the most s i m p l e a m i n o a c i d s -g l y c i ne, al ani ne, The l ast v a l i n e , l e u c i n e a n d i s o l e u c i ne. (Leu, lle, Val) contain three amino acids b ra n c h e d a l i p h a ti c s i d e c hai ns, hence thev are referred to as branched chain amino acids. 2. Hydroxyl group containing amino acids : Serine, threonine and tyrosine are h y d ro x y l g ro u p c o n ta i n i n ga mi no aci ds. Tyrosine-being aromatic in nature-is u u s u a l l yc o n s i d e re d n d e r a romati cami no acids. 3. Sulfur containing amino acids : Cysteine w i th s u l fh y d ry l g ro u p a n d methi oni ne with thioether group are the two amino acids incorporatedduring the course of protein synthesis. Cystine, another ami no aci d , is i mportant furcontai ni ng sul of formed by condensation two molecules of cysteine. 4. Acidic amino acids and their amides : Aspartic acid and glutamic acids are dicarboxylic monoamino acids while asparagi ne and gl utami ne are th eir resoective amide derivatives. All these distinct codons four amino acids possess for their incorporationinto proteins. 5. Basicamino acids : The three amino acids l ysi ne, argi ni ne (w i th guani di no group) and histidine (with imidazole ring) are dibasic monocarboxylic acids. They are highly basic in character. 6. Aromatic amino acids : Phenylalanine, tyrosineand tryptophan(with indole ring)
  • 54. 48 BIOGHEMISTF|Y are aromatic amino acids. Besides these, histidine may also be considered under this category. pyrrolidine 7. lmino acids: Prolinecontaining r ing is a u n i q u e a mi n o a c i d . l t h as an imino group (=NH), insteadof an amino group (-NH2) found in other amino acids. proline is an a-imino acid. Therefore, B. Classification of amino acids based on polarity : Amino acids are classified into 4 groups based on their polarity. The polarity in turn reflects the functionalrole of amino acids in protein structure. 1. Non-polar amino acids : These amino acids are also referredto as hydrophobic (water hating). They have no charge on t he ' R ' g ro u p . T h e a m i n o a c i d s i n c l uded in this group are - alanine, leucine, isoleucine, valine, methionine, phenylalanine, 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 participate in hydrogen bonding of protein structure. The simple amino acid glycine (where R = H) is also consideredin this category. The amino acids in this group areglycine, serine, threonine, cysteine, glutamine, asparagineand tyrosine. 1. Essentialor indispensableamino acids : The amino acids which cannot be synthesized hy the body and, therefore, need to be supplied through the diet are called essentialamino acids. They are proper Browth and required for maintenanceof the individual. The ten for amino acids listed below are essential humans (and also rats) : Arginine, Valine, Histidine, lsoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine,Tryptophan. lThe code A.U HILL, MP., T. T. (first letter of each amino acid) may be memorized to recall essential amino acids. Other useful codes are H. VITTAL, LMP; PH. VILLMA, TT, PW TIM HALL and MATTVILPhLy.I The two amino acids namely arginineand histidine can be synthesizedby adults and not by growing children, hencethese are considered as semi-essential amino acids (remember Ah, to recall). Thus, 8 essential while amino acids are absolutely 2 are semi-essential. 2. Non-essential or dispensabte amino '10 acids : The body can synthesizeabout amino acids to meet the biological needs, hence they need not be consumed in the diet. These are-glycine, alanine, serine, ne, asparagi glutamate, cysteine, aspartate, glutamine,tyrosineand proline. 3. Polar amino acids with positive 'R' group : D. Amino acid classification based on their The three amino acids lysine, arginine metabolic fate : The carbon skeleton of amino and histidine are included in this group. acids can serve as a precursor for the synthesis or or 4. Polar amino acids with negative'R'group : of glucose(glycogenic) fat (ketogenic) both. From metabolic view point, amino acids are The dicarboxylic monoamino acidsaspartic acid and glutamic acid are divided into three groups (for details, Refer Chapter lA. consideredin this group. C. Nutritional classification of amino acids : The twenty amino acids (Iable 4.1) are required for the synthesisof variety of proteins, besides other biological functions. However, all these 20 amino acids need not be taken in the diet. Based on the nutritionalrequirements, amino acids are grouped into two classes+ssential and nonessential. 1. Glycogenic amino acids : These amino acids can serve as precursors for the formation of glucose or glycogen. e.g. glycine,methionine etc. alanine,aspartate, 2. Ketogenic amino acids : Fat can be synthesizedfrom these amino acids. Two ami no aci ds l euci ne and l ysi ne are exclusivelyketogenic.
  • 55. Ghapter 4 : PFIOTE|NS AND AMTNO ACTDS 3. Glycogenic and ketogenic amino acids : T he fo u r a m i n o a c i d s i s o l e u c i n e, phenyl alanine, tryptophan, tyrosine are pre_ cursorsfor synthesis glucoseas well as of fat. Selenocysteine - the 2i st amino acid As already stated, 20 amino acids are commonly found in proteins.ln recent years,a 21s tam ino a c i d n a me l ys e l e n o c y s te i nh a sbeen e added. lt is found at the active sites of certain enzymes/proteins (selenoproteins). e.g. gluta_ thione peroxidase,glycine reductase,5,-deiodinase,thioredoxin reductase. Selenocysteine is an unus u a l a mi n o a c i d c o n ta i n i n g th e trace elem ents e l e n i u mi n p l a c e o f th e s u l fu r a tom of cysteine. z-!H-coo- -cH-coo- rinJ NHd Cysteine Selenocysteine 49 3. Taste: A mi no aci ds may be sw eet (C l y, Ala, Val), tasteless(Leu) or bitter (Arg, lle). Monosodium glutamate (MSC; ajinomoto) is used as a flavoring agent in food industrv,and Chinese foods 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 exceptglycine possess optical isomers due 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 -ami no aci ds i n compari sonw i th glyceraldehyde has been given (SeeFig.4.t). 5. Amino acids as ampholytes: Amino acids contai n both aci di c (-C OOH ) and basi c (-NH2) groups. They can donate a proton or accepta proton, henceamino acids are regarded as ampholytes. Zwitterion or dipolar ion : The name zwitter Incorporation of selenocysteine into the is derived from the German word which means proteinsduring translationis carried out by the codon namely UCA. lt is interesting note that hybrid. Zwitter ion (or dipolar ion) is a hybrid to UCA is normally a stop codon that terminates molecule containing positiveand negativeionic grouPs. protein biosynthesis. Another unique feature ts that selenocysteine enzymatically generated is The amino acids rarelyexist in a neutralform from serinedirectly on the tRNA (selenocvsteine- with free carboxylic (-COOH) and free amino IRNA), and then incorporatedinto proteins. (-N H 2) groups.In strongl yaci di c pH (l ow pH ), the amino acid is positively charged (cation) Pyrrolysine-the 22nd amino acid? : In tne w hi l e i n strongl y al kal i ne pH (hi gh pH ), i t i s year 2002, some researchers have describedyet negatively charged(anion).Eachamino acid has another amino acid namely pyrrolysineas the a characteri sti c pH (e.g. l euci ne, pH 6.0) at 22nd amino acid present in protein. The stop which it carries both positive and negative codon UAG can code for pyrrolysine. chargesand existsas zwitterion (Fig.a.Z. IsoelectricpH (symbol pl) is defined as the pH at which a molecule existsas a zwitterion or T he am i n o a c i d s d i ffe r i n th e i r p h ysi co- dipolar ion and carries no net charge. Thus, the c hem ic alpr o p e rti e s h i c h u l ti m a te l yd e te rmrne mol ecul e i s el ectri cal l y w neutral . the characteristics proteins. of The pl value can be calculatedby taking the pKa valuescorresponding the ionizable average to A. Physical propefiies groups. For instance,leucine has two ionizabre 1. Solubility: Most of the amino acids are groups/and its pl can be calculatedas follows. us uallys olu b l ei n w a te r a n d i n s o l u b l ei n o rgani c solvents. Properties of amino acids 2. M elt in g p o i n ts : Ami n o a c i d s g e n eral l y melt at higher temperatures, often above 200.C. a L p1=4!9.9 =6.s z
  • 56. 50 BIOCHEMISTFIY H I R -C -C O OH I NHz groups namely carboxyl (-COOH) group and ami no (-N H 2) group. Reactions due to -COOH Amino acid H I R- C- CO O H I, NHi Cation (lowpH) group 1. A mi no aci ds form sal ts(-C OON a) w i th (-COOR') with alcohors. basesand esters H I R-C-COOI NHz Anion (hish pH) 2. D ecarboxyl ati on:A mi no aci ds underg o decarboxylation producecorresponding to ami nes. R-CH-COO ----+R-CH2 + CO2 NHa NHT + Thi s reacti onassumes gni fi cancen th e si i l i vi ng cel l s due to the formati onof many biologically important amines. These i ncl ude hi stami ne, tyrami neand y-ami no butyric acid (CABA)from the amino acids histidine, tyrosine and glutamate, respectively. H I R-C-COOI NHi Zwitterion (isoelectric pH) 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 ), l e u c i n e i s fo u n d a s z w i tteri on.Thus t he pH o f th e m e d i u m d e te rm i n e sthe i oni c nat ur eo f a mi n o a c i d s . F or t h e c a l c u l a ti o n f p l o f a m i n o aci ds w i th o more than two ionizablegroups,the pKasfor all the groups have to be taken into account. Titration of amino acids : The existenceof differentionic forms of amino acids can be more easily understood by the titration curves. The graphic representationof leucine titration is depicted in Fi9.4.3.At low pH, leucine existsin a fully protonated form as cation. As the titration proceedswith NaOH, leucine loses its protons and at isoelectric pH (pl), it becomes a zwitterion. Further titration results in the formation of anionic form of leucine. S om e m o re d e ta i l s o n i s o e l e c tri cpH are discussed under the properties of proteins 1p. 60) . E, Chemica! properties The general reactions of am t no aci ds are mostly due to the presenceof two f uncti onal 3. Reaction with ammonia: The carboxyl group of dicarboxylic amino acids reacts with NH3 to form amide Aspartic acid + NH, ------;Asparagine Glutamic acid + NH. ------+ Clutamine 14 13 12 11 F-C H -C OO- I NHz I I pH 7 6 5 3 2 1 0 0.5 -+ 1.0 1.5 2.0 -=+ Eouivalents NaOH of Fig, 4.3 : Titrationcurue of an amino acid-leucine (R = (CH),-CH-CH,-; PK, = Dissociationconstant for COOH; pl = lsoelectric pH; pK, = Dissociationconstant for NHI).
  • 57. -d*-*d,h Ghapter 4 : PFIOTEINS AND AMINO ACIDS Reactionsdue to -NH2 group 4. The amino groups behave as bases and combine with acids (e.g. HCI) to form s a l ts(-N H i C l -). acidsareverfrp- nt for protein structure and functions. Selected examples hereunder. are given . Collagen-the most abundant protein in mammals-contains 4-hydroxyproline and 5. Reaction with ninhydrin: The cr-amino 5-hydroxylysine. acids react with ninhydrin to form a pu rp l e , b l u e o r p i n k c o l o u r compl ex . Histones-the proteins found in association (Ruhemann's purple). with DNA-contain many methylated, phosphorylated acetylatedamino acids. or Amino acid + Ninhydrin---+ Ketoacid + N H r+ C Oz + H y d r i ndanti n Hydrindantin NH: + Ninhydrin-----+ + R u h e ma nn' purpl e s Ninhydrin reaction is effectivelyused for the quantitativedeterminationof amino acids and proteins. (Nofe : Proline and hydroxyproline give yellow colour with ni n h y d ri n ). 6. Colour reactionsof amino acids : Amino acids can be identifiedby specificcolour reactions (See Table 4.3). . y-Carboxyglutamic acid is found in certain pl asmaprotei nsi nvol ved i n bl ood cl otti ng. . Cystine is formed by combination of two cysteines. Cystine is also considered as deri ved ami no aci d. B. Non-protein amino acids : These amino acids,although neverfound in proteins,perform several bi ol ogi cal l y i mportant functi ons. They may be either d-or non-cr-amino acids. A selected of theseamino acidsalong with their list functions is given in Table 4.2. 7. Transamination Transfer of 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. C. D-Amino acids : The vast majority of amino acids isolatedfrom animalsand olantsare of L-category.Certain D-amino acids are also found i n the anti bi oti cs (acti nomyci n-D, val i nomyci n, grami ci di n-S ). D -seri ne and D-aspartate are found in brain tissue. D8. Oxidative deamination: The amino acids Gl utami c aci d and D -al ani ne are present i n undergooxidativedeaminationto liberate bacteri alcel l w al l s. free ammonia (Refer Chapter l5). ]{ON.STANDARD AMINO ACIDS Amino acids usefu! as drugs Therea certainnon-standard amino acidsthat B es id e s th e 2 0 s ta n d a rd a mi n o aci ds are used as drugs. (described above) present in the protein structure,there are several other amino acids . D -P eni ci l l ami ne (D -di methyl gl yci ne), a whic h ar e b i o l o g i c a l l yi m p o rta n t. h e sei ncl ude T metabol i teof peni ci l l i n, i s empl oyed i n the the amino acid derivativesfound in proteins, chel ati ontherapyof W i l son' sdi sease. s i s Thi non- pr o te i n m i n o a c i d s p e rfo rmi n g peci al i zed a s possi bl e nce D -peni ci l l ami ne si can effecti vely f unc t ion sa n d th e D -a m i n o a c i d s . chelate copper. A. Amino acid derivatives in proteins : The . N-Acetylcysteine used in cystic fibrosis, is and 20 standard amino acids can be incoroorated chroni c renal i nsuffi ci encv, i t can functi on as into proteins due to the presenceof universal as an anti oxi dant. genetic code. Some of these amino acids undergo specific modification after the protein . Gabapentin (y-aminobutyrate linked to cvclohexane)is used as an anticonvulsant. svnthesisoccurs. These derivativesof amino r'
  • 58. 52 B IOC H E MIS TR Y Amino acids Function(s) L cr,-Amino acids Ornithine I I I Arginosuccinicl acid Citrulline Thyroxine I I J Triiodothyronine S-Adenosylmethionine Homocysteine Homoserine phenylalanine (DOPA) 3,4-Dihydrory Creatinine Ovothiol Azaserine ll. Non-s,-amino acids p-Alanine p-Aminoisobutyric acid yAminobutyric(GABA) acid (ALA) &Aminolevulinic acid Taurine Intermediates biosynthesis inthe ofurea. Thyroid hormones from derived tyrosine. system. Methyl inbiological donor for heart inmethionine metabolism. factor coronary A risk Intermediate diseases metabolisms. Intermediate inthreonine, aspartate methionine and pigment. serves tor A neurotransmitter, asa precursormelanin lrom and in Derived muscle excretedurine in eggs, acts Sulfur containing acid amino found fertilized and asan antioxidant Anantibiotic pantothenic and A Component ofvitamin acid coenzyme productpyrimidine metabolism. End of produced glutamic A neurotransmitter from acid (finally of Intermediate synthesisporphyrin heme) inthe Found association bile in with acids. polypeptide chains referredto as subunits.The spatial arrangement these subunits is known of quaternarystructure. as Proteins the polymersof L-cr-amino are acids. lThe structural hierarchy of proteins is The structure proteinsis rathercomplex which of of comparablewith the structure a building. The can be divided into 4 levels of organization amino acids may be consideredas the bricks, (Fig.4.4) : the twists in a the wall as the primary structure, 1. Primary structure: The linear sequence of wall as the secondarystructure,a full-fledged room as the tertiary structure.A amino acids forming the backbone of proteins self-contained (polypeptides). bui l di ng w i th si mi l ar and di ssi mi l arrooms w i ll be the quaternarystructurel. 2. Secondary structure: The spatial The term protein is generally used for a arrangement of protein by twisting of the polypeptide containing more than 50 amino poly pep ti d e h a i n . c acids. ln recent years, however, some authors 3. Tertiary structure: The three dimensional have been usi ng' pol ypepti de' even i f the structureof a functional orotein. They numberof ami no aci ds i s a few hundreds. prefer to use protein to an assembly of 4. Quaternary structure : Some of the proteins are composed of two or more polypeptidechains with quaternarystructure.
  • 59. Chapter 4 : PROTEINS AND AMINO ACIDS Primary structure Secondary structure 53 Tertiary structure Quaternary structure Fig. 4.4 : Diagram maticrepresentation proteinstructu of re (Note : Thefour subunitsof tuvo typesin quaternary structure). PRIMARY STRUCTUREOF PROTEIN double bond in character.lt generallyexists in trans configuration. Both -C=O and -NH Eachprotein has a unique sequence amino of groups of peptide bonds are polar and are ac ids w h i c h i s d e te rm i n e d b y th e genes involved in hydrogen bond formation. contained in DNA. The primary structureof a protein is largely responsible for its function. A Writing of peptide structures: Conventionally, vast majority of genetic diseasesare due to the peptidechainsare writtenwith the free amino abnormalitiesin the amino acid sequencesof end (N-terminalresidue) the left, and the free at proteins i.e. changes associatedwith primary carboxylend (C-terminal residue) the right.The at structureof protein. amino acid sequence readfrom N-terminalend is T he a m i n o a c i d c o mp o s i ti o n o f a protei n to C-terminal end. Incidentally, the protein also from the N-terminalamino determinesits physicaland chemical properties. biosynthesis starts aci d. Peptide bond The amino acidsare held togetherin a protein by covalent peptide bonds or linkages.These bonds are rather strong and serve as the cementing material between the individual amino acids (considered bricks). as Formation of a peptide bond: When the amino group of an amino acid combines with Ihe carboxyl group oI another amino acid, a peptide bond is formed (Fig.a.D. Note that a dipept id ew i l l h a v e tw o a mi n o a c i d s and one peptide (not two) bond. Peptides containing more than 10 amino acids (decapeptide)are referred to as polypeptides. Characteristics of peptide bonds: The peptide bond is rigid and planar with partial H +l H3N-C-COO'l + Rl Aminoacid 1 +l Fl"N-C-COOR2 Aminoacid2 Hzo HH +l I H3N-C-€O-l'iH-C-COOtl R1 R2 Dipeptide Fig.4.5 : Fomation of a peptide bond.
  • 60. B IOC H E MIS TR Y 54 Shorthand to read PePtides: The amino acids in a PePtideor Protein are representedby the 3-letter or one letter abbreviation. This is the chemical shorthandto write proteins. fruN--glrt"t"te--cysteine-glycine-CooE Glu _ C _G CYs - GIY Aminoacidsin a Peptide symbols Oneletter Threelettersymbols name Peptide Glutamyl cysteinYl glYcine Naming of peptides: For naming a in Fig.4.6 : lJseof symbols representing peptide peptides, the amino acid suffixes bondsis and twopeptide wrth3 aminoacids (Note: Atripeptide (glycine), -an (tryptophan),-afe -ine teft whilefree-COt is on the right)' shown;Free-NHtr is on the Glutamate) are changed to -Yl with amino the exception of C-terminal 2. Degradation of protein or polypeptide acid. Thus a tripeptide composed of an Ninto smaller fragments. terminal glutamate,a cysteineand a C-terminal g l u ta m y l -c y s te i n y l -gl yci ne. gly c in e i s c a l l e d of 3. Determination the amino acid sequence' ln the Fig.4.6,the naming and representation 1. Determination of amino acid composition of a tripeptideare shown. in a protein : The protein or polypeptide is to liberate the amino Dimensions of a PePtide chain : The completely hydrolysed quantitatively estimated' The dimensions of a fullv extended polypeptide acids which are carried out either by acid or chain are depicted in Fig.4.7.The two adjacent hydrolysismay be enzyme hydrolysis' atoms are placed at a distanceof 0.36 alkali treatment or by cr-carbon however results in nm. The interatomicdistancesand bond angles Treatment with enzymes, smaller peptidesrather than amino acids. are also shown in this figure. Pronase is a mixture of non-specific of primary stvueture Deterrnination proteolytic enzymes that causes complete The primary structure comprisesthe identi- hydrolysisof proteins. fication of constituentamino acids with regard Separation and estimation of amino acids: to their quality, quantity and sequence in a The mixture of amino acids liberatedby protein A protein structure. pure sample of a protein or hydrolysis can be determined by chromatofor the determination graphic techniques. The reader must refer a polypeptide is essential of primary structurewhich involves 3 stages: Chapter 41 tor the separation and quantitative Knowledge on n 1. D e te rm i n a ti o o f a mi n o a c i d composi ti on. determination of amino acids' Fig.4.7 : Dimensionsof a futly ertended polypeptide chain' (The-distancebetween two adiacent a-carbon atoms is A'36 nm)'
  • 61. Chapter 4 : PROTEINS AND AMINO ACIDS l JJ tFr OrN{' -/ -No, reagent Sanger's i prot"in tabetting i Hydrolysis i-+fr"" amino acids Edman'greagent i Proteinlabelling Hyorolysls i Hydrolysis ,$ Polypeptide (-N-terminal M) R I N-CH-COO- (DNP)Dlnltrophenyl amlnoacld I I + ldentifled by chromatography Phenylthlohydantoln (PTH)amino acld v I ldontlfledby chromatography Ftg. 4.8 : Sanger's reagent (llluoro 2,4-dinitrobenzene) and Edman's reagent (Phenyl isothiocyanate)in the determination of amino acid sequence of a protein (AA-Amino acid). primary structure proteins of will be incomplete withouta thorough understanding chromatoof graphy. (c) Breakdown of polypeptides into fragments: Polypeptides are degraded into smallerpeptidesby enzymaticor chemical methods. 2. Degradation protein into smallerfragof ments : Proteinis a large moleculewhich is Enzymatic cleavage Theproteolytic : enzymes polypeptide such as trypsin, chymotrypsin, sometimes composed individual of pepsin and chains.Separation polypeptides essential elastaseexhibit specificity in cleaving the of is beforedegradation. peptide bonds (Refer Fig.8.V. Among these enzymes/ trypsin is most commonlyused. lt (a) liberation of polypeptides:Treatment hydrolyses peptidebondscontaining the lysine with urea or guanidinehydrochloride or arginineon the carbonyl(-C:O) side of disrupts the non-covalent bonds and peptidelinkage. dissociates protein into polypeptide the units.For cleaving disulfide linkages the Chemical cleavage: Cyanogen bromide betweenthe polypeptide units,treatment (CNBr)is commonlyusedto split polypeptides with performicacid is necessary. into smallerfragments. CNBr specifically splits peptidebonds,the carbonylside of which is (b) Numberof polypeptides: number The of by polypeptide chainscan be identified by contributed the aminoacid methionine. treatmentof protein with dansylchloride. 3. Determination amino acid sequence of : It specifically binds with N-terminal amino The polypeptides their smaller or fragments are acidsto form dansylpolypeptides which conveniently utilizedfor the determination of on hydrolysisyield N-terminaldansyl sequence aminoacids. of Thisis donein a stepaminoacid.Thenumber dansvl of amino wise mannerto finally build up the order of acidsproduced equalto the number is of amino acids in a protein.Certainreagents are polypeptide chainsin a protein. (Fig,4,A. employed sequence for determination
  • 62. 55 ElIOCHEMISTRY chai n. B y anal ys ing reagent: Sangerused 1-fluoro2, ami no aci dsi n a pol ypepti de Sanger's (FDNB) determine DNA that codes for 4-dinitrobenzene to insulin the nucleotidesequenceof structure. FDNB specifically binds with protein, it is possibleto translatethe nucleotide N-terminal amino acid to form a dinitrophenyl sequence into amino acid sequence. This (DNP)derivative peptide. of This on hydrolysis technique,however,fails to identifythe disulfide yields DNP-amino that occur in the amino acids acid (N-terminal) free bondsand changes and aminoacidsfrom the restof the peptide chain. after the protein is synthesized(post-translational DNP-amino acid can be identified chromato- modifications). bv graphy. OF STRUCTURE PROTEIN reagenthas limited use since the SECONDARY Sanger's peptide chain is hydrolysed aminoacids. to The conformation of polypeptide chain by Edman's reagent: Phenyl isothiocyanateis the Edman'sreagent.lt reactswith the Nterminal aminoacid of peptide form a phenyl to thiocarbamyl On treatment with mild derivative. (PTH)-amino acid,phenylthiohydantoin acid,a cyclic compoundis liberated. This can be (Fig.a.A. identifiedby chromatography twisting or folding is referred to as secondary structure.The amino acids are located close to each other in their sequence. Two types of secondary structures, a-helix and p-sheef, are mai nl y i denti fi ed. lndian scientist Ramachandran made a significant contribution in understanding the of spatial arrangement polypeptidechains. Edman's reagent has an advantage since a peptide can be sequentially degraded liberating N-terminal aminoacidsone afteranother which u-l{elix can be identified. This is due to the factthatthe spiral structure a-Helixis themosfcommon peptideas a whole is not hydrolysed only but of of protein. lt has a rigid arrangement releases PTH-amino acid. polypeptide chain. a-Helical structurewas This is an automaticmachineto Sequenator: (1951) which is by and determinethe amino acid sequencein a proposed Pauling Corey regardedas one of the milestonesin the (with around 100 residues). is polypeptide lt research. The salientfeatures of basedon the principleof Edman's degradation biochemistry are s-helix (Fig.a.9 givenbelow (described above). Amino acidsare determined from N-terminalend. The PTHsequentially 1. The a-helix is a tightly packedcoiled amino acid liberatedis identifiedby high- structure extending with aminoacid sidechains (HPLC). outwardfrom the centralaxis. performanceliquid chromatography Sequenator takesabout 2 hoursto determine 2. The a-helix is stabilized by extensive eachaminoacid. H hydrogen bonding.lt is formedbetween atom to to attached peptideN, and O atom attached Overlapping peptides peptide The hydrogen bondsare individually C. ln the determination primarystructure of of to they enough (enzymatic chemical) weakbut collectively, arestrong protein, methods several or stabilize helix. the Thisresults the aresimultaneously employed. in peptides. is due to formation overlapping except firstand the of This bonds, 3. All the peptide the specific actionof different agents different last in a polypeptidechain, participatein on peptides hydrogen sitesin the polypeptide. Overlapping bonding. are very usefulin determining amino acid the 3.5 4. Eachturn of a-helixcontains amino sequence. of acids and travelsa distance 0.54 nm. The spacing eachaminoacid is 0.15 nm. of Reverse sequencing technique formed 5. a-Helix is a stableconformation It is the geneticmaterial(chemically DNA) with which ultimatelydetermines sequence the of spontaneously the lowestenergy.
  • 63. Chapten 4 : PR0TEINS AND AMINO ACIDS 57 Clu) or basic(Lys, Arg, His) aminoacidsalso interfere with o-helixstructure. p-Pleated sheet p This is the second (hence type of structure after a) proposed by Pauling and Corey. p-Pleated sheets (or simply p-sheets)are composed two or more segments fully of of extended peptide chains (Fig,4,10).ln the p-sheets, the hydrogen bonds are formed between the neighbouring segments of polypeptide chain(s). Parallel and anti.parallel p.sheets The polypeptide chainsin the p-sheets may be arrangedeither in parallel (the same (opposite direction)or anti-parallel direction). This is illustrated Fig.4,l0. in p-Pleatedsheet may be formed either by separate polypeptide chains (H-bonds are interchain) a single polypeptide or chainfolding backon to itself(H-bonds intrachain). are (A) Flg.4.9 : Diagrammatic representation secondary of structureof protein-a righthandeda-helix H I (l-lndicate -C-R groupsof aminoacids; (B) N-Terminal C-terminal dotted blue lines are hydrogen bonds; Note that only a few hydrogen bonds shown for clarity). (C) N-Terminal C-terminal C-Terminal # N-terminal 6. The right handedo-helix is more stable thanlefthanded helix(a righthanded helixturns in the direction thefingers righthandcurl that of when its thumbpointsin the direction helix the rises). F19.4.10 Structureof B-pleatedsheet(A) Hydrogen : polypeptidechains(B) Parallelp-sheet bondsbetween (C)AntiparallelB-sheet. (Note : Redctrctes A in 7. Certain proline) aminoacids(particularly disrupt a-helix.Large the number acidic(Asp, of represent emlnoacid skeleton-C-R l. I
  • 64. 58 B IOC H E MIS TR Y polypeptideswhich may be identical or "unrelated. are as Suchproteins termed oligomers quaternary The structure. individual and possess polypeptide chains are known as monomers, protomers or subunits.A dimer consitsol two polypeptides has while a tetramer four. Ft,.4.11 : Diagrammatic representation a protein of containing a-helixandB-pleatedsheet(blue). Bonds in quaternary structure: The by are monomeric subunits held together nonconvalent bonds namely hydrogen bonds, and hydrophobicinteractions ionic bonds. Importanceof oligomericproteins: These proteins play a significant in the regulation role and Occurrence of p-sheets: Many proteins of metabolism cellularfunction. As containp-pleated sheets. such,the cx-helix Examples oligomeric proteins: Hemoof and p-sheet commonlyfound in the same globin, are lactate aspartate transcarbomylase, protein structure(Fig.4.ll). In the globular dehydrogenase. p-sheets proteins, form the core structure. Other typesof secondary structures:Besides Bonds responsible for p-structures described above, the protein structure the cr-and p-bends and nonrepetitive(less organised is by Protein structure stabilized two typesof structures) secondary structures alsofoundin are and non-covalent. bonds-covalent proteins. : and 1. Covalent bonds Thepeptide disulfide TERTIARY STRUCTURE OF PROTEIN bondsare the strong bondsin proteinstructure. The three-dimensional arrangement of The formation of peptide bond and its have protein structure is referred to as tertiary chracteristics beendescribed. structure. lt is a compact structure with Disulfide bonds:A disulfide bond(-5-O is hydrophobic chains side heldinterior whilethe formedby the sulfhydryl groups(-SH) of two hydrophilicgroupsare on the surface the cysteine residues, to produce cystine of protein molecule.This type of arrangement Gig.a.l2A). The disulfide bondsmay be formed ensures stabilitv the molecule. of chain or between in a single polypeptide polypeptides. These bonds contribute to different Bonds of tertiary structure: Besidesthe the structuralconformationand stability of hydrogen bonds,disulfide bonds(-S-S), ionic interactions (electrostatic bonds) and proteins. hydrophobic interactions contribute the also to 2. Non-covalent bonds: There are,mainly, tertiarystructure proteins. of bonds. four typesof non-covalent Domains: The term domain is used to (a) Hydrogenbonds: The hydrogen bonds represent the basic units of protein structure of atoms areformedby sharing hydrogen (tertiary) function. polypeptide and A with 200 between the nitrogen and carbonyl amino acidsnormallyconsists two or more of oxygen of different peptide bonds domains. (Fig,4.12D. Eachhydrogen bond is weak A they are strong. large but collectively OUATERNARYSTRUCTUREOF PROTEIN bondssignificantly numberof hydrogen contribute the proteinstructure. to A greatmajorityof the proteins composed are of single polypeptidechains. Some of the proteins, however, consist of two or more (b) Hydrophobic side bonds: The non-polar aminoacidstendto be chainsof neutral
  • 65. AND AMINO ACIDS chapter 4 : PFIOTEINS 59 NH-CH-CO,,'V,^. gHz (A) I S I, q/sdne (- (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 induced dipoles. i CHz NH-CH-CO/'.:,,,, (B) r,.//^,,^_ C-CH - N ././././ ill ?R1 ! 'RaO -;-8,.^./././ /^,,,,^- N (c) NH- CH- CO,'^./,'," HC-CHs I CH^ lsoleucine '7 I' H33 I /,'v'r,'v,^NH(D) Leuclne COl"r,^r,^r,'. NH-CH-CO,,'.,,a..,, I Aspanate t He ,4/'.,/- " Lvslna ( HzhI NH-CH- CO,/././ positively of basic acidic amino acids with charged groups (e.g. -NHj) amino acids (Fi9.4.1 2D). Examples of protein structure Structure humaninsulin Insulinconsists of : of two polypeptide A chains, and B (Fig.a.lA, The A chain hasglycineat the N-terminal end and asparagine the C-terminal at end. The B chain has phenylalanine alanineat the and N- andC-terminal ends,respectively. Originally, insulin is synthesized a singlepolypeptide as preproinsulin which undergoes proteolytic processing give proinsulinand finally insulin. to The structural aspects hemoglobin of and colfagenare respectively given in Chaptersl0 and 22. Methods to determine protein structure For the determination secondaryand of tertiaryprotein structures, X-raycrystallography is most commonly used. Nuclear magnetic (NMR)spectra proteinsprovides resonance of structuraland functional informationon the atomsand groups present the proteins. in (A) Flg,4.12: Majorbonds protein in structure Dbultide bond (B) Hydrogen bonds(C) Hydrophic bonds 21 with each other in closely associated proteins(Fig.a.l2Q.As such, theseare not true bonds. The occurrenceof hydrophobic forces is observed in 1 aqueous environment wherein the molecules forcedto staytogether. are (c) Electrostatic bonds: These bonds are formed bv interactions between (e.g. groups negatively charged COO-)of ti SS ll 19 30 Bchaln Ftq.4.13: Diagrammatic representation of humaninsulin structu re
  • 66. 60 B IOGH E MIS TR Y Methods for the isolation and purification of proteins P epsi n-' | .1; asei n-4.6; uman al bumi n-4. 7; C H Urease-S.0; Hemoglobin-6.7 Lysozyme-l .0. 1 ; 5. Acidic and basic proteins: Proteins in Severalmethodsare employed to isolateand which the ratio (e Lys + e Ard/(e Clu + e Asp) it purify proteins.Initially,proteinsare fractionated by using differentconcentrations ammonium greater than 1 are referred to as basic proteins. of sulfate or sodium sulfate. Protein fractionation For acidic proteins,the ratio is lessthan 1 . may also be carried out by ultracentrifugation. 6. Precipitationof proteins: Proteins exist in Protein separation is achieved by utilizing col l oi dal sol uti on due to hydrati on of pol a r can be electrophoresis, isoelectricfocussing, immuno- groups(-C OO-, -N H t, -OH ). P rotei ns of electrophoresis, ion-exchangechromatography, precipitatedby dehydrationor neutralization gel-filtration, high performance liquid chromato- polar groups. graphy(HPLC) etc. The detailsof thesetechniques Precipitationat pl : The proteins in general are describedin Chapter 4l. are least soluble at isoelectric pH. Certain proteins(e.9.casein) easilyprecipitated get when the pH is adjusted to pl (4.6 lor casein). Formati onof curd from mi l k i s a marvel l ous 1. S o l u b i l i ty: Pro te i n s fo rm col l oi dal example of slow precipitationof milk protein, solutionsinsteadof true solutionsin water. This casein at pl. This occurs due to the lactic acid is due to huge size of protein molecules. produced by fermentation of bacteria which 2. Molecular weight : The proteins vary in lowers the pH to the pl of casein. t heir m o l e c u l a r w e i g h ts , w h i c h , i n turn, i s Precipitationby salting out: The processof depend e n t o n th e n u mb e r o f a mi no aci d protein precipitationby the additionalof neutral r es idue s . Ea c h a mi n o a c i d o n a n average salts such as ammonium sulfate or sodium sulfate contributesto a molecularweight of about 1 10. i s know n as sal ti ng out. Thi s phenomenonis Majority of proteinsholypeptides may be explained on the basisof dehydration of protein composed of 40 to 4,000 amino acids with a proteinmolecules salts. by This causes increased molecular weight ranging from 4,000 to protein interaction, resulting in molecular 440,00O.A few proteins with their molecular aggregation and precipitation. weights are listed below : The amount of salt required for protein fns u l n -5 ,7 0 0 My o g l o b i n -1 ,7 H emogl obi i ; OO; n- preci pi tati ondepends on the si ze (mol ecul ar 64,450; Serum albumin-69,000. weighg of the protein molecule. In general,the 3. S h a p e : T h e re i s a w i d e v a ri a ti on i n the higher is the protein molecularweight, the lower Thus, serum pr ot eins h a p e .l t m a y b e g l o b u l a r(i n sul i n), oval is the salt requiredfor precipitation. globulins are precipitated by half saturation with (albumin)fibrous or elongated(fibrinogen). ammonium sulfatewhile albumin is precipitated 4. lsoelectric pH : lsoelectric pH (pl) as a by full saturation. Salting out procedure is propertyof amino acids has been described. The convenientlyusedfor separating serumalbumins nat ur e o f th e a m i n o a c i d s (p a rti c ul arl ythei r from gl obul i ns. ionizablegroups)determines the pl of a protein. The addition of small quantities of neutral T he ac i d i c a mi n o a c i d s (As p , C l u ) and basi c salts increasesthe solubility of proteins. This amino acids (His, Lys,Arg) stronglyinfluencethe pl. At isoelectric pH, the proteins exist as process called as nlting rn is due to the interaction low salt at zwitterions or dipolar ions. They are electrically diminishedprotein-protein concentration. neutral (do not migratein the electricfield) with m inim u m s o l u b i l i ty , ma x i mu m p re ci pi tabi l i ty Precipitation by salts of heavy metals : Heavy and least buffering capacity. The isoelectric metal ions like Pb2+, Hg2+, Fe2+, Zn2+t Cd2+ pH(pl) for some proteinsare given here cause precipitation of proteins. These metals P RO P E R T IES OF P R OT E IN S
  • 67. 51 Ghapter 4 : PFIOTEINS AND AMINO ACIDS being positivelycharged,when added to protein charged)in alkalinemedium solution(negatively resultsin precipitateformation. Precipitation by anionic or alkaloid reagents: Proteinscan be precipitatedby trichloroacetic aci ac id, s u l p h o s a l i c y l ia c i d , p h o s p h otungsti c d, c pic r ic a c i d , ta n n i c a c i d , p h o s p h o mol ybdi c d aci etc. By the addition of these acids, the proteins existing as cations are precipitatedby the anionic form of acids to produce proteinsulphosalicylate, protein-tungstate, proteinpicrate etc. Precipitation by organic solvents: Organic solvents such as alcohol are good protein precipitatingagents.They dehydratethe protein molecule by removing the water envelope and cause precipitation. 7. Colour reactions of proteins : The proteins give several colour reactions which are often useful to identify the nature of the amino acids presentin them. Biuret reaction : Biuretis a compoundformed by heating urea to 180"C. NHe tC :O 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. o tl tl o The presenceof magnesiumand ammonium ions interfere in the biuret test. This can be overcome by using excessalkali. given by proteinsdue to The colour reactions the presence specificamino acids are given in of Table 4,3. These reactions are often useful to know the Dresence absenceof the said amino or aci ds i n the gi ven protei n. DENATURATION The phenomenon of disorganization of native protein structure is known as denaturation. Denaturation results in the loss of secondary, tertiaryand quaternary structure proteins. This of i nvol ves a change i n physi cal , chemi cal a nd biological propertiesof protein molecules. NH C=O NHz Reaction Specificgroup or amino acid 1, Biuret reaction When biuret is treated with dilute copper s ulf a tei n a l k a l i n e m e d i u m, a p u rpl e col our i s obtained. This is the basis of biuret test widely used for identificationof proteinsand peptides. Biuret test is answered by compounds c ont a i n i n gtw o o r mo re C O-N H groups i .e., peptide bonds. All proteins and peptides possessing least two peptide linkages i.e., at tripeptides (with 3 amino acids) give positive biuret test. Histidine is the only amino acid that answersbiuret test.The principle of biuret test is conveniently used to detect the presence of pr ot e i n si n b i o l o g i c a lfl u i d s . T h e m echani sm of peptide Two linkages 2. Ninhydrin reaction a-Amino acids 3. Xanthoproteic reaction Benzene ofaromatic ring amino (Phe, Trp) acids Tyr, 4. Milllons reaction group Phenolic (Tyr) 5. Hopkins-Cole reaction Indole (Trp) ring 6. Sakaguchi reaction group Guanidino (Arg) (Cys) 7, Nitroprussidereaction Sulfhydrylgroups 8. Sulfur test (Cys) Sulfhydrylgroups 9. Pauly's test lmidazole (His) ring groups (Tyr) Phenolic
  • 68. BIOCHEMISTFIY 62 Denaturation . Nativeprotein Fiq.4.14 : Denaturationof a protein. bonds to enzymes. Cooking causes protein denaturation and, therefore, cooked food Heat, violent shaking, (protein) is more easily digested. Agents of denaturation Physical agents: X-ravs,UV radiation. 8. Denaturation is usually irreversible.For Chemical agents : Acids, alkalies, organic instance,omelet can be preparedfrom an egg solvents(ether, alcohol), salts of heavy metals (protein-albumin) the reversal not possible. is but (Pb, Hg), urea, salicylate. 9. Careful denaturation is sometimesreversible (known as renaturation). HemoSlobin of denaturation Gharacteristics undergoes denaturation in the presence of 1. The native helical structureof protein is salicylate.By removal of salicylate,hemoglobin lost (Fig.4.l4). is renatured. 2. The primary structure of a protein with 10. Denaturedprotein cannot be crystallized. peptide linkages remains intact i.e., peptide Coagulation : The term 'coagulum' refersto a bonds are not hydrolysed. semi-solid viscous precipitate of protein. 3. The protein loses its biological activity. lrreversibledenaturationresults in coagulation. 4. Denatured protein becomes insoluble in Coagulation is optimum and requires lowest t he s olv en ti n w h i c h i t w a s o ri g i n a l l ys o l ubl e. temperature at isoelectric pH. Albumins and globulins (to a lesser extent) are coagulable 5 The viscosity ol denatured protein proteins. Heat coagulation test is commonly (solution) increaseswhile its surface tension used to detect the presence of albumin in urine. decreases. Flocculation: lt is the process of protein in with increase is 6. Denaturation associated pH. The precipitateis precipitationat isoelectric ionizable and sulfhydrylgroups of protein. This referredto as flocculum. Casein (milk protein) is due to loss of hydrogenand disulfide bonds. can be easily precipitated when adjusted to 7. Denaturedprotein is more easily digested. isoelectric pH (4.6 by dilute acetic acid. This is due to increasedexposure of peptide Flocculation is reversible. On application of
  • 69. 63 AND AMINOACIDS PROTEINS heat, flocculum can be converted into an ir r ev er s i b l e s s ,c o a g u l u m . ma CLA S S IF IC A T IO N OF P R OT E INS Proteinsare classifiedin severalways. Three proteinsbasedon their major typesof classifying f unc t ion , c h e m i c a l n a tu re a n d sol ubi l i ty properties and nutritional importance are here. discussed 7. Genetic proteins: Nucleoproteins. 8. Defenseproteins: Snakevenoms, lmmunogl obul i ns. 9. Receptorproteins for hormones,viruses. Thi s i s a more comprehensi ve and popul ar cl assi fi cati on orotei ns. l t i s based on tne of : ! i ti ami no aci d composi ti on,structure,shape and sol ubi l i ty properti es. P rotei ns are broadly Basedon the functionsthey perform,proteins cl assi fi ed nto 3 maj or B roups i ar e c las s i fi e di n to th e fo l l o w i n g g ro ups (w i th 1 . Simple proteins: They are composed of ex am pl e s ) only amino acid residues. 1. Structural proteins : Keratin of hair and 2. Conjugated proteins: Besidesthe amino nails ,c o l l a g e no f b o n e . aci ds, these protei ns contai n a non-protein 2. Enzymes catalyticproteins: Hexokinase, or moiety known as prosthetic group or pepsr n. group. conj ugati ng 3. T ra n s p o rt p ro te i n s : H e mo g l o b i n, serum 3. Derived proteins: Theseare the denatured album in . or degradedproductsof simple and conjugated 4. H o rmo n a l p ro te i n s : In s u l i n, grow th oroterns. nor m on e . The above three classes are further 5. Contractile proteins: Actin, myosin. subdi vi dedi nto di fferentgroups.The summar y of protein classification given in the Table4.4. is 6. S to ra g ep ro te i n s : O v a l b u mi n ,gl utel i n. 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 diuersilied such os enzymes, hormones, clotting factors, immunoglobulins, storage proteins 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 required proportion by the human body e.g. egg olbumin. Cooking resultsin protein denaturotion exposingmore peptide bondsfor easydigestion. Monosodium glutamate (MSG) ts used os a flauoring agent in loods to increasetaste and flauour. ln some indiuidualsintolerant to MSG, Chinese restaurantsyndrome (brief and reuersiblellu-like symptoms)is obserued.
  • 70. 64 BIOCHEMISTF|Y Scleroproteins Albumins Globulins Glutelins Prolamines Histones Gollagens Elastins Keratins Nucleoproteins Glycoproteins Mucoproteins Lipoproteins Phosphoproteins Chromoproteins Metalloproleins Coagulated prot€ins Proteoses Peptones Proteans Metaproteins Polypeptides Peptides Globins Protamines l. Simpleproteins (a) Globular proteins: These spherical are or oval in shape, solublein wateror other solvents digestible. and (i) Albumins: Soluble t n water and dilute salt solutions and coagulated by heat. e.g. serum albumin, (e8d,lactalbumin (milk). ovalbumin (ii) Globulins:Solublein neutraland dilute salt solutions e.g. serum (eggyolk). globulins, vitelline (iii) Glutelins: Soluble dilute in acids and alkaliesand mostlyfound in plants (rice). e.g. glutelin(wheat), oryzenin (iv) Prolamines: Soluble 7O1"alcohol in e.g. gliadin(wheat), zein (maize). (v) Histones: Strongly basic proteins, solublein waterand diluteacidsbut insoluble diluteammonium in hydroxide e.g.thymushistones, histones of codfishsperm. (vi) Globins Theseare generally : consideredalongwith histones. However, globins not basicproteins are are and not precipitated NH/OH. by (vii) Protamines: They are strongly basic and resemblehistonesbut smaller in size and soluble in NH4OH. Protamines are also found in association with nucleic acids e.g. sperm proteins. (b) Fibrous proteins : These are fiber like in shape,insoluble in water and resistant to digestion,Albuminoids or scleroproteins constitutethe most predominantgroup of fibrous proteins. (i) Collagens are connective tissue proteins lacking tryptophan. Collagens,on boiling with water or di l ute aci ds, yi el d gel ati n w hi ch i s soluble and digestible. (ii) Elastins:These proteinsare found in elastic tissues such as tendons and arteries. (iii) Keratins: These are present in structures e.g. hair, nails, exoskeletal horns.Human hair keratincontainsas much as 14% cystei ne. 2. Coniugated proteins (a) Nucleoproteins: Nucleic acid (DNA or RNA) is the prostheticBroup e.g. nucleones. histones,nucleoprotami (b) Glycoproteins The prosthetic group is : carbohydrate, which is less than 4"/" of protein, The term mucoprotein is used if the carbohydratecontent is more than 4o/o. ovomucoid(eggwhite). e.g. mucin (saliva),
  • 71. Chapter 4 : PROTEINS AND AMINO ACIDS 65 (c) Lipoproteins pointof in (described Fromthe nutritional : Protein found already). with lipidsas the prosthetic view, proteinsare classified into 3 categories. combination groupe.g.serumlipoproteins, membrane 1. Completeproteins Theseproteinshave : lipoproteins. all the ten essential aminoacidsin the required (d) Phosphoproteins: by Phosphoric acid is the proportion the humanbody to promotegood prosthetic group e.g. casein (milk), growth.e.g. egg albumin,milk casein. vitelline(eggyolk). pro2. Partiatly proteins:These incomplete teinsare partially lacking one or moreessential (e) Chromoproteins: prosthetic groupis The coloured in nature e.g. hemoglobins, amino acidsand hencecan promotemoderate growth.e.g. wheat and rice proteins(limiting cytochromes. Lys,ThO. (0 Metalloproteins proteins metal : These contain 3. Incomplete proteins: These proteins ions suchas Fe,Co, Zn, Cu, Mg etc., e.g. lack one or more essential amino (Zn). completely (Cu), anhydrase ceruloplasmin carbonic growthat all acids.Hencethey do not promote 3. Derived proteins : The derived proteins e.g. gelatin (lacks Trp),zein (lacks Trp, Lys). are of two types.The primaryderived are the denaturedor coagulated first hydrolysed BIOTOGICALLY IMPORTANT PEPTIDES or products proteins. secondary The derived are of Severalpeptides occur in the living orgathe degraded(due to breakdownof peptide nisms that display a wide spectrumof bioproducts proteins. of bonds) logical functions. is Generally, term'peptide' the (a) Primaryderivedproteins appliedwhen the numberof constituent amino of (i) Coagulated proteins: Theseare the acids is lessthan 10. Someexamples biologically activepeptides and theirfunctions are denatured proteins produced by described here. agentssuch as heat, acids,alkalies coagulated 1. Glutathionelt is a tripeptide etc. e.g.cookedproteins, : composed of (eggwhite). albumin glutathione y3 amino acids.Chemically, is glutamyl-cysteinyl-glycine. lt is widelydistributed (ii) Proteans: These are the earliest in nature and exists in reducedor oxidized productsof protein hydrolysisby states. etc. enzymes,dilute acids,alkalies whichareinsoluble water. fibrin in e.g. 2G-SH =+ G=S-S-G formed from fibrinogen. Reduced Oxidized (iii) N,lgf6plsteinsTheseare the second : stageproductsof protein hydrolysid with slightly obtainedby treatment e.g. acid acidsand alkalies stronger and alkalimetaproteins. Functions: In a steady state, the cells generallymaintaina ratio of about 100/1 of CSH to G-S-S-C. The reversible oxidationreduction glutathione important manyof is for of its biological functions. (b) Secondary : are derivedproteins These the . progressive of hydrolyticproducts protein hydrolysis. These include proteoses, polypeptides peptides. peptones, and r G. Nutritional classification of proteins is The nutritive valueof proteins determined amino acids of by the composition essential Clutathione as for serves a coenzyme certain enzymes PCE,synthetase, e.B.prostaglandin glyoxylase. lt prevents the oxidation of sulfhydryl (-SH) groups of several proteins to (-S-S-1 groups. disulfide Thisis essential for the protein function, including that of enzymes.
  • 72. B IOC H E MIS TFIY 66 It is believed that glutathione in association participates the with glutathionereductase in formationof correctdisulfidebonds in several proteins. (a peroxi dase sel eni umcontai ni ng gl utathi one enzyme). 2 GSH+ tjrorl99l!!9!5G - s - s - G + 2 H2o 2. Thyrotropin releasing hormone (IRFI) : lt Clut at h i o n e (re d u c e d ) p e rfo rm s s p eci al i zed TRH is a tripeptide secretedby hypothalamus. functions in erythrocytes stlmulatespituitary gland to releasethyrotropic ( i) lt ma i n ta i n s BC m e m b ra n es tru cture R and normone. integrity. 3. Oxytoci n: l t i s a hormone secretedby (ii) lt protects hemoglobin from getting posteri orpi tui tarygl and and contai ns9 ami no contraction Oxytocin causes acids(nonapeptide). oxidized by agentssuch as HzOz. of uterus. Clut ath i o n e i s i n v o l v e d i n th e tra nsoortof 4. Vasopressin(antidiuretic hormone, ADI{) t am ino a c i d s i n th e i n te s ti n e a n d ki dney producedby posterior y-glutamyl cycle or Meister cycle ADH is also a nonapeptide tubules via pi tui tary gl and. l t sti mul ates dneys to retai n ki (Refer Chapter 8). the blood pressure. water and thus increases G lut ath i o n e i s i n v o l v e d i n th e d e t oxi cati on I A 5. A ngi otensi ns: ngi otensi n i s a decapepprocess. The toxic substances (organoti de (10 ami no aci ds) w hi ch i s converted to phosphates, nitro compounds)are converted angi otensi nl l (8 ami no aci ds). The l ater has t o m er c a p tu ri c c i d s . a more hypertensive effect. Angiotensin ll also Toxic amounts of peroxidesand free radicals stimulates the release of aldosterone from produced in the cells are scavanged by adrenalgl and. EIOMEtrICAL / CLIIhIICALCONCEPTE sa Collagen is the most abundant protein in mammols. lt is rich in hydroxyproline and hydroxylysine. Seuerolbiologicolly important peptides are known in the liuing orgonism.Theseinclude glutathione t'or the maintenonce of 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. lg Antibiotics such os actinomycin, gramicidin, bocitracin and tyrocidin are peptide in nature. 6 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 ocids ol biological importonce are known. These include thyroxine ornithine, citrulline and arginosuccinic acid (intermediotesol urea synthesis), 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 using protein precipitating agents such os phosphotungstlc acid ond trichloroaceticacid. of Heot coagulationtest is mosf commonly employedto detect the pre.sence albumin in urine.
  • 73. PFOTEINS AND AMINOACIDS 67 6. Methionine enkephalin: lt is a pentapeptide found in the brain and has opiate like f unc t ion . l t i n h i b i tsth e s e n s eo f a p a i n. 9. Aspartame: lt is a dipeptide (aspartylphenyl al ani ne methyl ester), produceo by a combi nati on of asparti c aci d and A 7. Bradykinin and kallidin : They are nona- phenyl al ani ne. spartamei s about 200 ti mes sweeter than sucrose, and is used as a and decapeptides, respectively. Both of them act i as powerful vasodilators.They are produced l ow -cal ori e arti f ci al sw eetner i n softdri nk i ndustry. from plasma proteinsby snakevenom enzymes. 10. Gastrointestinal hormones: 8. Peptide antibiotics: Antibiotics such as Castrin, gr am id i n ,b a c i tra c i nty ro c i dn a n d a c tinomyci n secretin etc. are the gastrointestinal ic , i peptides w hi ch serveas hormones. are peptide in nature. I. Protelns are nltrogen containing, most abunddnt organlc mscromolecules wtdely distributed in animals ond plants. They perform structurol and dynamic lunctions in 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. Selenocysteine hss been recently identified as the 27st amlno acld, and is found ln certain protelns. 3. Amino ocidspossesstwo functional groups namely carboxyl(-CooH) qnd amlno (-NH). ln the physiologlcolsystem,they exist as dipolar ions commonly relerred to os zwitteriois. 4. Besidesthe 20 standard omino acidspresent in proteins, there are seuerolnon-stondard amino ocids. These include the omino acid deriuatlues found in proteins (e.g. hydroxyprollne, hydroxylysine)ond, non-protein amino acids (e.g. ornithine, citrulltne). 5. The structure of protein is dluided into t'our leuels of organizatlon. The primary structure represents the linear sequence of amino ocids. The twisting ond spatial arrangement of polypepttde chdin is the secondary structure. Tertiary structure constltutes the three dimensional structure of a t'unctional protein. The assemblyof slmilar or dtssimilar polypepilde subunils comprlses quaternary structure. 5. The determlnation of primary structure of a protein inuoluesthe knowledgeol quality, quantity and the sequenceof amino acids in the polypeptide. Chemical and enzymatic methods are employed t'or the determinotion of primary structure. 7. The secondary structure of protein mainly conslstsof a-helix anilor ftsheet. a-Helix is stabiltzedby extensive hydrogen bondlng. ftPleated sheet is composedol two or more segmentsof fully extended polypepttde choins. 8. The tertlory and quaternary structures ol protetn are stabiltzed by non-coualent bonds such os hydrogen bonds, hydrophobic interactions, ionic bonds etc. 9' Protelns are 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.
  • 74. 68 B IOC H E MIS TR Y I. Essayquestions 1. Describe the classification amino acids along with their structures. of of 2. Discuss organization proteinstructure. the of Cive an accountof the determination primary structure orotein. of 3. Describe the classification proteins of with suitable examples. 4. Write an accountof non-standard amino acids. 5. Discuss the importantbiologically activepeptides. II. Short notes (a) Essential amino acids, (b) Zwitterion,(c) Peptidebond, (d) Edman'sreagent,(e) a-Helix, (j) (fl p-Pleated (h) point, (i) Clutathione, Quaternary structure sheet,(g) Denaturation, lsoelectric of protein. IIL Fill in the blanks 1. The averagenitrogencontent of proteins 2. Proteins the polymers -. are of 3. Name the sulfurcontaining essential amino acid 4. The chargedmoleculewhich is electrically neutralis known as -. 5. The non -o amino acid present coenzyme -. in A 6. The bondsformingthe backbone proteinstructure of 7. The amino acid that is completely of destroyed acid hydrolysis protein by L The numberof peptidebonds present a decapeptide in -. 9. The chemical name of Sanger's reagent 10. The phenomenon disorganization nativeproteinstructure known as -. is of of IV.Multiple choice questions 11, T h e i mi n o a c i d fo u n d i n p ro tei nstructure (a )A rg i n i n e ) Pro l i n e ) H i sti di ne Lysi ne, (b (c (d) 12, T h e fo l l o w i n gi s a n o n -p ro te in no aci d ami (a) Ornithine(b) Homocysteine Histamine All of them. (c) (d) 13. The bonds in proteinstructure that are not brokenon denaturation. (a) Hydrogenbonds(b) Peptidebonds(c) lonic bond (d) Disulfidebonds. 14. Sequenator an automatic is chain. machine determine amino acid sequence a polypeptide in to The reagent used in sequenator is (a) Sanger's (b) reagent CNBr (c) Trypsin(d) Edman's reaBent, 15. The reactiongiven by two or more peptidelinkages is (a) Biurettest (b) Ninhydrintest (c) Xanthoproteic reaction(d) Pauley's test.
  • 75. nNucXefcAcids nNucXeotfldes and here are two types of nucleic acids, genes control the protein synthesis through the I namely deoxyrihonucleic acid (DNA) and mediation of RNA, as shown below ribonucleicacid (RNA). Primarily,nucleic acids A ----+ RNA-----| Prlteir of serve as repositories and transmitters genetic inf or m at i o n . The interrelationship these three classes of of (DNA, RNA and proteins) biomolecules constitutes Brief history the cenfral dogma of molecular biology or more commonly the central dogma of life. DNA w a s d i s c o v e re d i n 1 8 6 9 b y Johann F r iedr ic h M i e s c h e r, a Sw i s s re s e a rcher.The C omponents of nucl ei e aei ds dem ons t ra ti o n th a t D N A c o n ta i n e d geneti c Nucleic acids are the polymers of nucleotides informationwas first made in 1944, by Avery, (polynucleotides) held by 3' and 5' phosphate Macleod and MacCarv. bridges.In other words, nucleic acids are built up by the monomeric units-nucleotides (lt may F unc t ion s o f n u c l e i c a c i d s be recalled that protein is a polymer of amino aci ds). DNA i s th e c h e m i c a l b a s i s o f h e re di ty and nay be regardedas the reservebank of genetic re for r r or m at i o n .D N A i s e x c l u s i v e l y s p o nsi bl e -aintaining the identity of different speciesof Nucleotides composedof a nitrogenous every are c ' ganis m s v e r m i l l i o n so f y e a rs .F u rther, o a sugarand a phosphate. Nucleoas oec t c e l l u l a rfu n c ti o ni s u n d e rth e controlof base, pentose of a of in f,{. The DNA is organized into geneg the tidesperform wide variety functions the besides the blocks or :urdamental units of genetic information. The livingcells, being building J 69
  • 76. 70 B IOC H E MIS TR Y (C) the pyrimidine cytosine isfoundin bothDNA and RNA. However,the nucleic acids differ with respectto the secondpyrimidinebase. DNA contains thymine (T) whereas RNA contains uracil (U). As is observed in the Fig,5.2, thymine and uracildifferin structure by (in (in the presence T) or absence U) of a methyl 8roup. Tautomeric fcrms of purines an€i pyriffiidines Flg.5.l : Generalstructureof nitrogenbases (A) Purine(B) Pyrimidine (Th€ pasltions nambercd are awrding to the intemetif'.'d'l systern). The existenceof a molecule in a keto (lactam) and enol (lactim) form is known as The tautomerism. heterocyclic rings of purines /o and pyrimidines with oto LC-/ functional m onome ri cu n i ts i n th e n u c l e i c a c i d (D N A and groups exhibittautomerism simplified as below. RNA) structure. These include their role as OH OH I structural components of some coenzymes of l. 'v-r-^1, .i - I -C = N B - c om p l e xv i ta m i n s (e .9 . F A D , N A D+ ), i n the Lactamform Lactlmform energy reactions of cells (ATP is the energy currency), and in the control of metabolic o reactions. STRUCTURE OF NUCLEOTIDES As already stated, the nucleotide essentially consistsof nucleobase, sugar and phosphate. (A) Adenlne Thetermnucleoside refers base sugar. to + Thus, (6-aminopurine) nucleotide nucleoside phosphate. is + H (G) Guanlne (2-amino 6-orypurine) Purines and pyrinnidines The nitrogenous bases found in nucleotides (and, therefore,nucleic acids) are aromatic heterocyclic compounds.The basesare of two types-purinesand pyrimidines. Their general structures depictedin Fig.S.l Purines are . are numbered the anticlockwise in directionwhile pyrimidines are numberedin the clockwise direction. And this is an internationally accepted system represent structure bases. to the of Major bases in nucleie acids The structures of major purines and pyrimidines foundin nucleic acidsareshownin Fig.5.2. DNA and RNAcontain same purines the namelyadenine(A) and guanine(C). Further, I Cytoslne(C) (2-ory4-aminopyrimidine) oo H3 HN" tI lll f*/ Hil (T) Thymine Uracil(U) (2,A-dioxy-Smethylpyrimidine) (2,4-dioxypyrimidine) purines(A, G) and Flg.5.2 : Structures major of pyrimidlnes T, U) foundin nucleicacids. (C,
  • 77. Ghapter 5 : NUCLEIC ACIDSAND NUCLEOTIDES NHe t- NHe N2-c N?cc t- a lll lll i/- 6"'"*"-" ^ I H Lactamform ,t-' HO OHH D-2-Deoryribose D-Ribose Lactim form Fig. 5.3 : The tautomeric forms of cytosine. Fig. 5.5 : Structures sugarspresentin nucleic acids of (riboseis foundin RNAand deoxyribose DNA;Note in the slructuraldifference Cz). at T he p u ri n e -g u a n i n e a n d p y ri m i di nes- Sugars of nucleic acids cytosine, thymine and uracil exhibittautomerism. (pentoses) The five carbon monosaccharides The lactam and lactim forms of cytosine are are found in the nucleic acid structure. RNA representedin Fi9.5.3. contains D-ribose while DNA contains At physiologicalpH, the lactam (keto)tauto- D-deoxyrihose. Ribose and deoxyribosediffer in meric forms are predominantlypresent. structure C2. Deoxyribose at has one oxygen less at C2 compared to ribose (Fig.s.A. Minor basesfound in nucleic acids : Besides the basesdescribed above, several minor and unusualbases are often found in DNA and RNA. These include 5-methylcytosine, Na-acetylcytosine, N6-methyladenine,N6, N0-dimethyladenine,pseudouracil etc. lt is believedthat the unus ualba s e si n n u c l e i c a c i d s w i l l h e l p i n the recognitionof specific enzymes. Other biologically important bases : The basessuch as hypoxanthine,xanthine and uric acid (Fig.5.4)are present in the free state in the cells. The former two are the intermediates rn pur ine s y n th e s i sw h i l e u ri c a c i d i s th e end product of purine degradation. Nomenclattrre of nueleotides The addition of a pentose sugar to base produces a nucleoside. lf the sugar is ribose, ribonucleosides are formed. Adenosine, guanosi ne, cyti di ne and uri di ne are the ribonucleosides A, C, C and U respectively. of lf the sugar is a deoxyribose, deoxyribonucleosides are produced. The term mononucleotide is used when a single phosphate moiety is added to a nucl eosi de. Thus adenosi ne monophosphate (AMP) contains adenine + ribose + phosphate. Purine basesof plants : Plantscontain certain The principal bases, their respective methylated purines which are of pharmacological interest. These include caffeine (of nucl eosi des and nucl eoti des found i n the of coffee), theophylline (of tea) and theobromine structure nucleic acids are given in Tahle5.1. Note that the prefix 'd' is used to indicate if the (of cocoa). sugar is deoxyribose(e.g. dAMP). The binding components Hypoxanthine Xanthine Uricacid (6-orypurine) (2,6-diorypurine) (2,6,8-trioxypurine) Fig. 5.4 : Structures of some biologically impoftant purines. of nucleotide The atoms i n the puri ne ri ng are .l numbered as to 9 and for pyrimidine as 1 to 6 (SeeFig.S.l).The carbonsof sugars are represented with (1 an associated prime for differentiation. Thus the pentose carbons are 1' to 5'.
  • 78. 72 BIOCHEMISTFIY Ribonucleoside Ribonucleotide (5'-monophosphate) Abbreviation (A) Adenine Adenosine Adenosine 5'-monophosphate or adenylate AMP (G) Guanine Guanosine Guanosine 5'-monophosphate or guanylate GMP (C) Cytosine Cytidine Cytidine Samonophosphate or cytidylate CMP (U) Uracil Uridine Uridine 5'-monophosphate or uridylate UMP Deoxyribonucleoside Deoxyribonucleotide (5'-monophosphate) Abbreviation (A) Adenine Deoxyadenosine Deoxyadenosine 5'-monophosphate or deoryadenylate dAMP (G) Guanine Deoxyguanosine Deoryguanosine Slmonophosphate or deoxyguanylate dGMP (C) Cytosine Deorycytidine Deorycytidine 5'-monophosphate or deoxycytidylate (T) Thymine Deoxythymidine Deoxythymidine 5'-monophosphate or deoxythymidylate dTMP The pentosesare bound to nitrogenousbases by p-N-glycosidic bonds.The Ne of a purine ring binds with C1111of a pentose sugar to form a covalent bond in the purine nucleoside.ln case of pyrimidine nucleosides, glycosidiclinkage the is between Nl of a pyrimidine and C'1 of a pentose. The hydroxyl groups of adenosine are esterified with phosphatesto produce 5'- or 3'-monophosphates. 5'-Hydroxyl is the most commonly esterified, hence 5' is usuallyomitted while writing nucleotide names. Thus AMP dCMP represents adenosine 5'-monophosphate. However, for adenosine 3'-monophosphate,the abbreviation3'-AMP is used. The structuresof two selected nucleotioes namefy AMP and TMP are depicted in Fig.5.6. H{urr:"freoside di- and triphosphates Nucleoside monophosphates possess only one phosphate moiety (AMP,TMP).The addition of second or third phosphates the nucleoside to 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 : The structures of adenosine S'-monophosphate(AMP) and thymidineS'-monophosphate(TMP) [*-Addition of second or third phosphate gives adenosine diphosphate (ADP) and adenosine triphosphate (ATP) respectivetyl.
  • 79. 73 ACIDSAND NUCLEOTIDES Chapter 5 : NUCLEIC 5. Arabinosylcytosine being used in cancer is therapy as it interferes with DNA replication. 6. The drugs employed in the treatmentof AfDS namely zidovudine or AZT (3-azido 2',3' -dideoxythym i ne) and d idanosne (dideoxyid i inosine) are sugar modified synthetic nucleotide analogs(For their structureand more details Refer Chapter 3A. 8-Azaguanine Fig. 5.7 : Structures of selected purine and pyimidine analogs. DNA is a polymer of deoxyribonucleotides (or simply deoxynucleotides). is composedof lt monomeric units namely deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxy(dTMP) The anionic properties of nucleotides and cytidylate(dCMP)and deoxythymidylate nucleic acids are due to the negative charges (lt may be noted here that some authors prefer to contributedby phosphategroups. use TMP for deoxythymidylate, since it is found only in DNA). The details of the nucleotide P UR|NE , P YR IM T D T N E structure are given above. AND NUCLEOTIDEANALOGS It is possible to alter heterocyclic ring or sugar moiety, and produce synthetic analogs of pur in e s , p y ri mi d i n e s , n u c l e o s i des and nucleotides.Some of the syntheticanalogs are T highly us e fu li n c l i n i c a l m e d i c i n e . h e structures of selectedpurine and pyrimidine analogs are given in Fi9.5.7. Schematic representation of polynucleotides The monomericdeoxvnucleotides DNA are in hefd together by 3',5'-phosphodiester bridges (Fi9.5.81. DNA (or RNA) structure is often represented a short-hand in form. The horizontal line indicatesthe carbon chain of sugar with The pharmacologicalapplicationsof certain base attached to C,,. Near the middle of the analogsare listed below horizontalIine is C3, phosphatelinkagewhile at the other end of the line is C5, phosphate linkage 1. Allopurinol is used in the treatment of (Fig.s.A. hyperuricemia and gout (For details, Refer Chapter l7). Ghargaff's rule of DNA composltion 2. S-Fluorouracil, 6-mercaptopurine, 8-azaErwin Chargaff in late 1940s quantitatively guanine, 3 -d e o x y u ri d i n e ,5 - o r 6 -a zauri di ne, analysed the DNA hydrolysatesfrom different 5- or 6-azacytidine and 5-idouracilare employed in the treatmentof cancers.These compounds species.He observedthat in all the specieshe get incorporated into DNA and block cell studied,DNA had equal numbersof adenineand thymine residues(A = T) and equal numbersof or olif er a ti o n . guanine and cytosine residues(G = C). This is 3. Azathioprine (which gets degraded to known as Chargaff's rule of molar equivalence suppress 6-mercaptopurine) is used to between the purines and pyrimidines in DNA i m munological rejection du ring transplantation. structure. The significance Chargaff's of rule was for the not immediatelv realised. The double helical is used 4. Arabinosyladenine treatment of neurological disease, viral structure of DNA derives its strength from Chargaff'srule (discussed later). enc eoha l i ti s .
  • 80. 74 B IOC H E MIS TFIY J I DNA structure is considered as a milestone in the era of modern biology. The structure of DNA double helix is comparable to a twisted ladder. The salient features of Watson-Crick model of DNA (now known as B-Df.lA) are described next (Fi9.5.9). 'end o I I ,: I o I I L 5' Hzr 2q' I {K 4 1' H uanrne OH I n-D-/l- C 3'end A Fig. 5.8 : Structureof a polydeoryribonucleotide segmentheld by phosphodiester bonds.On the lower part is the representation shorthad formof ol oligonucleotides. | u ==:: DNA, and RNAs which are Single-stranded usually single-stranded, not obey Chargaff's do rule. However, double-stranded RNA which is the genetic material in certain viruses satisfies Chargaff'srule. A ____T A_ _ _ _ u T ---- A J = =:='J DNA DOUBLE HELIX The double helical structure of DNA was proposed by lames Watson and Francis Crick in 1953 ( N o b e l Pri z e , 1 9 6 2 ). T h e e l u ci dati onof Fig.5.9 : (A) Watson-Crick model of DNA helix (B) Complementary base pairing in DNA helix.
  • 81. Ghapter 5 : NUCLEIC ACIDSAND NUCLEOTIDES 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 w hi l e G-C pai r has 3 hydrog en bonds.The G = C is strongerby about 50% than A = T. H (B) .Z-=.,-N.-,, l' ttt'o tl n. . 8. The hydrogen bonds are formed between a puri ne and a pyri mi di ne onl y. l f tw o puri n es face each other, they would not fit into the allowable space. And two pyrimidines would be too far to form hydrogen bonds. The only base arrangement possible in DNA structure, from spatialconsiderations A-T, T-A, G-C and is c-c. 9. The complementarybase pairing in DNA helix proves Chargaffs rule. The content of adenine equals to that of thymine (A = T) and guanine equals to that of cytosine (G = C). Fiq.5.10 : Complementarybase paiing in DNA (A) Thymine pairs with adenine by 2 hydrogen bonds (B) Cytosine pairs with guanine by 3 hydrogen bonds. 1 . Th e D N A i s a ri g h t h a n d e dd o u bl e hel i x. l t consists ol two polydeoxyribonucleotide chains (strands) twisted around each other on a c om m o n a x ts . 2. The two strands are antiparallel, i.e., one strand runs in the 5' to 3' direction while the ot her in 3 ' to 5 ' d i re c ti o n . T h i s i s c o mparabl e to two parallel adjacent roads carrying traffic in opposite direction. 10. The genetic information resideson one of the two strands known as template strand or sense strand. The opposite strand is antisense strand. The double helix has (wide) major grooves and (narrow) minor grooves along the phosphodiester backbone.Proteinsinteractwith DNA at these grooves,without disrupting the base pairs and double helix. Sonformations 0f DNA double helEx Variation in the conformation of the nucleotides of DNA is associated with conformational variants of DNA. The double helical structure of DNA exists in at least 6 different forms-A to E and Z. Among these, B, A 3. The width (or diameter)of a double helix and Z forms are important (Table 5.2). The is 20 A o (2 n m). B-form of DNA double helix, described bv Watson and Crick (discussed above),is the most 4. Each turn (pitch) of the helix is 34 A" form under physiological (3.4 nm) with 10 pairs of nucleotides, pair predominant each conditions. Each turn of the B-form has 10 base placed at a distanceof about 3.4 Ao. pai rs spanni nga di stance 3.4 nm. The w i dth of 5. Each strand of DNA has a hydrophilic of the doubl e hel i x i s 2 nm. phosphate deoxyribose backbone(3'-5' phosphoThe A-form is also a right-handedhelix. lt diester bonds) on the outside (periphery) the of contai ns11 basepai rsper turn. Therei s a ti l ti ng m olec u l e w h i l e th e h y d ro p h o b i c bases are of the base pairs by 2O" away from the central stackedinside (core). axts. 6. The two polynucleotide chains are not The Z-form (Z-DNA) is a left-handed helix identical but complementaryto each other due and contains '12 base pairs per turn. The to base pairing.
  • 82. 76 BIOCHEMISTFIY Feature Helix type B-DNA A-DNA Z.DNA Right-handedRight-handedLetl-handed Triple-stranded Helical (nm) diameter 1.84 per Distance each complete turn(nm) 3.4 3.2 4.5 Riseperbase pair(nm) 0.34 0.29 0.37 Number base ol pairs complete per rurn 10 Basepairtilt Certain antitumor drugs (e.g. cisplatin) produce bent structurein DNA. Such changed structure can take up proteins that damage the DNA. +1 9" l1 -1.2' (variable) tz DNA Triple-strandedDNA formation may occur due to additional hydrogen bonds between the form two Thus, a thymine can selectively bases. Hoogsteen hydrogen bonds to the adenine of A-T pair to form I-A-L Likewise, a protonated cytosinecan also form two hydrogenbonds with guanine of C-C pairs that resultsin C-G-C. An outline of Hoogsteentriple helix is depicted in Fig.5.11. -9" Triple-helical structure is less stable than double helix. This is due to the fact that the three grooveThrough groove negatively charged backbone strands in triple Helix rotation Major base Minor axis pairs (variable) helix results in an increased electrostatic repul si on. polynucleotide strands of DNA move in a somewhat 'zig zag' fashion, hence the name Z - DNA . It is believed that transition between different helical forms of DNA plays a significantrole in regulatinggene expression. Four-stranded DNA l with very high contents of Polynucleotides guanine can form a novel tetrameric structure 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 molecular structures are important for recognition of DNA by proteins and enzymes. This is in fact neededfor the DNA to discharge its functions in an appropriate manner. Some selected unusual structures of DNA are brieflv described. Eent DNA I n gen e ra l , a d e n i n e b a s e c o n ta i n ing D N A tractsare rigid and straight.Bentconformationof DNA occurs when A-tracts are replaced by other basesor a collapse of the helix into the minor groove of A-tract. Bendingin DNA structurehas also been reported due to photochemical damage or mispairingof bases. Fig" 5.11 : An outline of Hoogsteen triple helical structure of DNA.
  • 83. 77 ACIDSAND NUCLEOTIDES chapter 5 : NUCLEIC C-tetraplexeshave been implicated in the called G-quarfefs. These structures are planar and are connected by Hoogsteen hydrogen recombi nati onof i mmunogl obul i ngenes, and bonds (Fig.S.12A). Antiparallel four-stranded in dimerization of double-strandedgenomic DNA structures, referred to as G-tetraplexes R N A of the human i mmunodefi ci encyvi rus (Hlu . have also been reported Gig.5.12Rl. namely The ends of eukarvoticchromosomes THE SIZE OF DNA MOLECULE telomeresare rich in guanine, and thereforeform -UNITS OF LENGTH In C-tetraplexes. recent years, telomeres have chemotherapies. for becomethe targets anticancer D N A mol ecul es are huge i n si ze. On an average, a pair of B-DNA with a thickness of 0.34 nm has a mol ecul ar w ei ght of 660 daltons. For the measurement lengths,DNA doubleof strandedstructureis considered, and expresssed in the form of base pairs (bp). A kilobase pair (kb) is 103 bp, and a megahasepair (Mb) is 106 pai bp and a gi gabase r (C b) i s 10e bp. The kb, Mb and Cb relations mav be summarized as fol l ow s : 1 kb = 1000 bp 1 Mb = 1000 kb = 1,000,000bp 1 C b = 1000 Mb = 1,000,000,000 bp s', (B) / f_l I ?-f ?-? II tt G-G I u G I u I G _G G_ G tl tl tl tl tl 3' s', t -l 3' The length of DNA varies from species to in species, and is usuallyexpressed termsof base pair composition and contour length. Contour length represents total lengthof the genomic the D N A i n a cel l . S omeexampl es organi sms i th of w bp and contour lengthsare listed. . l , phage vi rus- 4.8 x 104 bp-contour l ength 16.5 mm. r: I G _G It may be noted here that the lengthsof RNA mol ecul es (l i ke D N A mol ecul es) cannot be expressedin bp, since most of the RNAs are single-stranded. s', (A) DNAstructure Parallel Fig.5.12: Four-stranded G4uartets (B) AntiparallelG-tetraplex. E. coli 1.5 mm. 4.6 x 106 bp - contour length D i pl oi d human cel l (46 chromosomes) 6.0 x 10e bp-contour l ength2 meters. It may be noted that the genomic DNA size is usual l y much l arger the si ze of the cel l or nucl euscontai ni ngi t. For i nstance,n humans, i a 2-meter long DNA is packed compactly in a nucleus of about 1Opm diameter.
  • 84. 78 B IOC H E MIS TtrIY T he ge n o m i c D N A rn a y e x i s t i n l i near or c ir c ular f o rm s . M o s t D N A s i n b a c teri a exi st as c los e d c i rc l e s . T h i s i n c l u d e s the D N A of bac t e ri a l c h ro mo s o m e s a n d th e extrac hr om os o m a lD N A o f p l a s mi d s .Mi to chondri a an< lc hlor o p l a s ts f e u k a ry o ti c e l l s a l s o contai n o c c ir c ular D N A. - Denaturation . R"a"tr-rti* Chr omo s o m a D N As i n h i g h e ro rg a n i sms l are m os t ly lin e a r. i n d i i z i d u a ih u rn a n c h ro m osomes Two strands c ont ain a s i n q l e D N ,t r-n o l e c u l e i th vari abl e w separated s iz es c om p a c tl y p a c k e d . T h u s th e smal l est c hr om os o mec o n ta i n s3 4 M b w h i l e th e l arsesr Fiq.5.13 : Diagrammaticrepresentationof denaturation and renaturationof DNA. one has 26 3 M b . ffiffiWATUffiAT[ON MF MN& STffiANffiS T he t r , v o s tra n d s o f D N A h e l i x a re hel d t oget her b y h y d ro g e n b o n d s . D i s ru pti on of hy c J r ogeb o n d s(b y c h a n g ei n p l -1 r i n creasen n o i iem per at u re ) re s u l ts i n th e s e p a rati on of poly nuc le o ti d e tra n c l s .h i s p h e n o n re o n of Joss s T n of helical structure of DNA is kno',vn as denaturatian (Fi9.5.1 3). The phosp[64;"r*"t bonds ar e n o t b ro k e n b y d e n a tu ra ti o n Lossof . helic al s t ru c tu rec a n b e m e a s u re db y increase in abs orb a n c e a t 2 6 0 n m i i n a sD ectrophotometer). Melting temperature (Im) is defined as the temperature w hi ch hal f of the hel i calstructure at of D N A i s l ost. S i nce C -C base pai rs are more stable(due to 3 hydrogenbonds)than A-T base pai rs (2 hydrogenbonds),the Tm i s greaterfor D N A s i vi th hi gherC -C content.Thus,the Tm i s 65" C for 35% C -C contentw hi l e i t i s 70' C for 5A % C -C content. Formanri de destabi l i zes hydrogen bonds of base pai rs and, therefore, l ow ers Trn.Thi schemi cal compoundi s effecti vel l , used i n recombi nant N A experi ments. D EIOMEDICAL CLINTCAL CONCEPTS / L€ D/VA is the reseruebank of genetic int'ormation,ultimately responsible t'or the chemical bcsis o/ life and heredity. Gt DNA is organized into genes, the t'undamental units of genetic int'ormation. Genes control protein biosynfhesis through the mediation of RNA. u-F Nucleic acids are the polymers of nucleotides.Certoin nucleotidesserue as B-complex uitamin coenzymes(EAD' IVAD+, CoA), carriers ot' high energy intermediates(UDP-glucose, S-adenosylmethionine) and secondmessengers hormonal action (cAME cGMP). ol Na Uric ocid is a purine, ond the end product ol purine metabolism, that has been implicated in the disordergout. Certain purine basesfrom plants such os caft'eine(oj coffee), theophylline (of tea) and theobromine (of cocoo) are of pharmacological interest. c'Ji' Synthetic analogs of bases(5-fluorouracil, 6-mercaptopurine.6-azauridine)are used to inhibit the growth of cancer cells. ut Certain antitumor drugs (e.g. cisplatin) can producebent DNA structureand damageit.
  • 85. Ghapter 5 : NUCLEIC ACIDSAND NUCLEOTIDES 79 Renaturation(or reannealing)is the process These 30-nm fibers are further organized into in which the separatedcomplementary DNA loops by anchoringthe fiber at M-rich regions namely scaffold-associated strandscan form a double helix. regions (SARS) a to protein scafold.During the courseof mitosis,the loops are further coiled, the chromosomes condenseand becomevi si bl e. As alreadv stated.the double-stranded DNA helix in e a c h c h ro m o s o meh a s a l e n g th that i s thousands times the diameterof the nucleus.For ins t anc e ,i n h u ma n s , a 2 -me te r l o n g D N A i s packed in a nucleus of about 'l0 pm diameter! T his is m a d e p o s s i b l e b y a c o mpact and p , of m ar v ello u s a c k a g i n ga n d o rg a n i z a ti o n D N A ins ide in c e l l . OrganEzation of prakaryotFc Dl{A I n pr ok a ry o ti c e l l s ,th e D N A i s o rg a ni zed c as a single chromosome in the form of a doublestranded circle. These bacterial chromosomes are packed in the form of nucleoids, by interaction with oroteins and certain cations ( poly am i n e s ). R N A i s a pol ymer of ri bonucl eoti des d hel together by 3',5'-phosphodiester bridges. A l thoughR N A has certai nsi mi l ari ti es i th D N A w structure,they have specific differences l . P entose: The sugar i n R N A i s ri bose i n contrastto deoxyribosein DNA. 2. P yri mi di ne: R N A contai nsthe pyri mi di ne uraci l i n pl ace of thymi ne (i n D N A ). 3. S i ngl e strand : R N A i s usual l y a si ngl estranded polynucleotide. However, this strand may fol d at certai n pl aces to gi ve a doubl estrandedstructure,if complementarybase pairs are i n cl ose proxi mi ty. 4. Chargaff's rule-not obeyed : Due to the single-strandednature, there is no specific rel ati on betw een puri ne and pyri mi di ne In the eukaryoticcells, the DNA is associated contents. Thus the guani necontenti s not equal with various proteins to form chromatin which to cytosi ne(as i s the case i n D N A ). then gets organized info compact structures namely chromosomes (Fig.SJa" 5. Susceptibilityto alkali hydrolysis : Alkali can hydrolyseRNA to 2',3'-cyclic diesters. This T he DN A d o u b l e h e l i x i s w ra p p e da roundthe is possible due to the presenceof a hydroxyl which are basic in core proteinsnamely histones group at 2' position. DNA cannot be subjected nature.The core is composedof two molecules to alkali hydrolysisdue to lack of this group. of histones(H2A, H2B, H3 and H4). Each core with two turns of DNA wrapped round it 6. Orcinol colour reaction : RNAs can be (approximatelywith 150 bp) is termed as a hi stol ogi cal l y i denti fi ed by orci nol col our nucleosome, the basic unit of chromatin. reaction due to the presenceof ribose. Nucleosomesare separatedby spacer DNA to which histone H1 is attached (Fig.S.l5). This TYPES OF RNA representing continuous string of nucleosomes, The three major types of RNAs with their stringform of chromatin is termed as beads-on-a respecti ve l ul arcomposi ti on cel are gi ven bel ow 10 nm fi b e r. T h e l e n g th o f th e DN A i s re c ons idera b l y d u c e db y th e fo rma ti o nof 10 nm 1. MessengerRNA (mRNA) : 5-1O"/" f iber . T h i s 1 0 -n m fi b e r i s fu rth e r c oi l ed to 2. TransferRNA (IRNA) : 10-200/" or oduc e 3 0 -n m fi b e r w h i c h h a s a sol enoi d 3. RibosomalRNA (rRNA) : 50-80% structure with six nucleosomesin everv turn. Srganization of eukaryotic DhlA
  • 86. 80 BIOCHEMISTF|Y Naked DNA doublehelix Jzn' 'Beads-on-a-string' formof chromatin I,.", 30-nmchromatin fibrecomposed of nucteosomes Chromosome an in extended form (non-condensed loops) Condensedform ol cnromosome Metaphase chromosome ,o*'', | Fig.5.l4 : Organization eukaryotic of DNAstructure theformof chromatin chromosomes. in and + I 11nm lnbmucleosome Fig. 5.15 : Structurcof nucleosomes.
  • 87. Ghapter 5 : NUCLEIC ACIDSAND NUCLEOTIDES Type of RNA Abbreviation Messenger RNA 81 Funclion(s) genelic Transfers intormation genes from to Ii99.9_0,.T.99. .p$giLL !9_$ltgli'e 9J. ... lt_qt:r.qg:l.:.qtE..t lf9l99.t..EllL Transfer RNA Ribosomal RNA Small nuclear RNA Small nucleolar RNA rRNA snRNA snoBNA Small cytoplasmic RNA Transfer-messenger RNA TMRNA scRNA Besidesthe three RNAs referred above, other RNAsare also presentin the cells.Theseinclude heterogeneousnuclear RNA (hnRNA), small nuc lear R N A (s n R N A), s m a l l n u c l e o l ar R N A (snoRNA)and small cytoplasmicRNA (scRNA). The major functionsof these RNAs are given in Table 5.3. Serves precursor mRNA other as for and RNAs. Transfers amino to mRNA protein acid for biosynthesis. Provides structural framework ribosomes. for processing. Involved mRNA in Plays keyrolein theprocessing rRNA a of molecules. Involvedtheselectionoroteins exoort. in of for present bacteria. short Mostly peptide in Adds tags proteins facilitate degradation t0 to the of proteins. incorrectly synthesized nucleotides) which is known as poly (A) tail. This tail may provide stabilityto mRNA, besides preventing it from the attack of 3'-exonucleases. mRNA molecules often contai certain modified bases such as 6-methyladenylates in the internal structure. The RNAsare synthesized from DNA, and are Transfer RNA (tRNAl primarily involved in the process of protein Transfer RNA (soluhle RNA) molecule biosynthesis (Chapter 2fl. The RNAs vary in their structureand function. A brief description contains 71-80 nucleotides(mostly 75) with a molecular weight of about 25,000. There are at on the major RNAs is given. least 20 speciesof tRNAs, corresponding 20 to Messenger amino acids present in protein structure. The RNA (mRNAl structure of tRNA (for alanine) was first The mRNA is synthesized the nucleus (in in elucidatedby Holley. eukaryotes) as heterogeneous nuclear RNA (hnRNA). hnRNA, on processing,liberatesthe The structure of IRNA, depicted in Fig.S.t6, that of a clover leaf. IRNA contains functional mRNA which entersthe cytoplasmto resembles participate in protein synthesis.mRNA has high mainly four arms, each arm with a base paired stem. molecular weight with a short half-life. 1. The acceptor arm : This arm is capped The eukaryotic mRNA is capped at the S'-terminal end C by 7-methylguanosine w i th a sequence C A (5' to 3' ). The ami no aci d triphosphate. is believedthat this cap helps to is attached to the acceptor arm. lt prevent the hydrolysis of mRNA by 5'-exo2. The anticodon arm : This arm, with the nucleases. Further, the cap may be also involved three specific nucleotide bases (anticodon), is in the recognitionof mRNA for proteinsynthesis. responsible for the recognitionof triplet codon The 3'-terminal end of mRNA contains of mRNA. The codon and anticodon are a polymer of adenylate residues (20-250 complementaryto each other. t
  • 88. 82 BIOCHEMISTRY el g-Amino acid I +A r -!, Acceptor arm D arm Complementary basepairs TrC arm Variable arm Anticodon arm f,ibosomal The ribosomes are the factories of protein synthesis. The eukaryotic ribosomes are major nucleoprotein composed of two complexes-60Ssubunit and 40S subunit. The 605 subunit contains 28S rRNA, 55 rRNA and 5.8S rR N A w hi l e the 40S subuni tcontai ns18S rRNA. The function of rRNAs in ribosomes not is clearly known. lt is believed that they play a si gni fi cant rol e i n the bi ndi ng of mR N A to ribosomesand protein synthesis. Other Fiq.5.16: Structureof transferRNA. 3. The D arm : lt is so named due to the presenceof dihydrouridine. RNA (rRNAl RNAs The various other RNAs and their functions are summarisedin Table 5.3. CATALYTIG RNAs-RI BOZYMES In certain instances, RNA componentof a the ribonucleoprotein (RNA irr association with 4. The TYC arm : This arm contains a protein) is catalytically active. Such RNAs are sequence of T, pseudouridine(represented by termedas ribozymes. leastfive distinctspecies At ps i, Y ) an d C . of RNA that act as catalysts have been identified. 5. The variable arm : This arm is the most Three are involved in the self processing v ar iable i n tR N A. Ba s e d o n th i s v a ri abi l i W , reactions of RNAs while the other two tRNAs are classifiedinto 2 categories: are regarded as true catalysts (RNase P and (a) Class I tRNAs : The most predominant rR N A ). (about 75"/") form with 3-5 base pairs Ribonuclease P (RNase P) is a ribozyme len g th " containing protein and RNA component. lt (b) Classll tRNAs : They contain 13-20 base cleaves IRNA precursors to generate mature pai r l o n g a rm. tR N A mol ecul es. Base pairs in tRNA : The structureof IRNA is RNA molecules are known to adapt maintained due to the complementary base just like proteins(i.e. enzymes). tertiarystructure pairing in the arms. The four arms with their The specific conformation of RNA may be respective base pairs are given below responsiblefor its function as biocatalyst. lt The acceptor arm - 7 bp is believed that ribozymes (RNAs) were The TYC arm - 5 bp functioning as catalysts before the occurrence The anticodon arm - 5 bp of protein enzymes, during the course of T h e D a rm evolution. -4 b p
  • 89. Shapten 5 : NUCLEIC ACIDS AND NUCLEOTIDES l. DNA is the chemical basisof heredity organized into genes, the basic units of genetic inJormotion. 2. RNAs ImFNA, fRNA and rRNA) are produced by DNA which in turn carry out protein synfhesis. 3. Nucleic acids are the polymers of nucleotides (polynucleoiides)held by 3' and 5' phosphodiester bridges. A nucleotide essentiolly consists of base + sugdr (nucleoside) and phosphate. 4. Besidesbeing the constituentsof nucleic acid structurc, nucleotidesperform a wide uariety ot' cellulor functians (e.9. energy carriers, metabolic regulators,second messengers etc.) (A) 5. Both DNA ond RNA contain the parines-adenine and guanlne (G)ond the pyrimidinecytosine(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-ribose in RNA. 6. The structure o/ DNA is a double helix (Watson-Crick model) composed ol two tutistedaround each othen The strandsare antiparollel sfronds ol polydeoxynucleotides held together by 2 or 3 hydrogen bonds t'ormed between the basesi.e. A = T: G : C. DNA sfructure satisfiesChargoff's rule that the content of A is equal to T, and that ol G equal to C. 7. Besides the double helical structure, DNA olso exisfs fn certain unusuol structuresbent DNA, triple-strand DNA, four-strand DNA. 8. RNA is usually a single stranded polyribonucleotide. mRNA is capped ot 5'terminol end by 7-methylGTP while at the 3'-terminal end, it contains o poly A toil. mRNA speciliesthe sequenceol amino scids in protein synfhesis. 9. 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. 10. Certain RNAs that mn function os enzymes are termed os ribozymes. Ribozymes were probablVfunctioningos cofolysfsbefore the occurrenceof protein enzymesduring ewlution. 83
  • 90. 84 BIOCHEMISTFIY I. Essayquestions 1. Describe the structure DNA. of 2. Name differentRNAsand discuss their structure. 3. Write an accountof structure, of functionand nomenclature nucleotides. presentin nucleic acids.Add a note on tautomerism. 4. Describe structure nitrogenous the of bases 5. "The backbone nucleicacid structure 3'-5'phosphodiester of is bridge."-justify. II. Short notes (a) Chargaff's (c) rule, (b) Riboseand deoxyribose, Hydrogenbonds in DNA, (d) Nucleoside, (e) Differentforms of DNA, (f) Transfer base RNA, (g) Purine basesof plants,(h) Complementary (j) pairs,(i) DNA denaturation, hnRNA. III. Fill in the blanks 1 . The fundamental unit of geneticinformation known as is 2 . DNA controlsproteinsynthesis throughthe mediation of 3 . Nucleic acidsare the polymers of 4 . The pyrimidinepresent DNA but absentin RNA in 5 . Ribose and deoxvribose differ in their structure aroundcarbonatom 6 . Nucleotideis composed of 7 . The scientist who observed that there existsa relationship betweenthe contentsof purinesand pyrimidines DNA structure = T; C = C) (A in B. The basepair G-C is more stableand stronger than A-T due to 9. Underphysiological condition,the DNA structure predominantly the form in is 10. The acceptor arm of IRNA contains cappednucleotide sequence a IV. Multiple choice questions 1'l. The nitrogenous basenot present DNA structure in (a) Adenine(b) Cuanine(c) Cytosine Uracil. (d) 12. The numberof basepairspresent each turn (pitch)of B-formof DNA helix in (a ) e (b ) 1 0 (c ) 1 1 (d ) 1 2 . 13. The backbone nucleicacid structure constructed of is by (a) Peptide (d) bonds(b) Glycosidicbonds(c) Phosphodiester bridges All of them. 14. The following coenzymeis a nucleotide (a) FAD (b) NAD+ (c) CoASH(d) All of them. 15. The nucleotide that serves an intermediate biosynthetic as for reaction (a) UDP-glucose CDP-acylglycerol S-Adenosylmethionin8 AII of them. (b) (d) (c)
  • 91. The etrzytnes spetrr. : "We are the catalystsof the liuing worU! Protein in nature, and in d.ctionspecifc, rapid and accurate; Huge in size but with srnall actiae centres; Highly erploited for disease diagnosisin hb cennel" In the laboratory,hydrolysisof proteinsby a strong acid at 100'C takes at least a couple of days. The same protein is fully digestedby the enzymes in gastrointestinal tract at body temperature(37'C) within a couple of hours. This remarkable difference in the chemical The student-teacher relationship may be a ' reactionstaking place in the living system is good example to understand how a catalyst exclusivelydue to enzymes.The very existence works. The students often find it difficult to learn of life is unimaginablewithout the presenceof from a text-book on their own. The teacher enzvmes. explainsthe subjectto the students and increases their understanding capability. lt is no wonder that certain difficult things which the students take daystogetherto understand, and sometimes do not understand all - are easily learnt under at the guidance of the teacher. Here, the teacher Berzeliusin 1836 coined the term catalysis ac t s like a c a ta l y s t i n e n h a n ci ng the (Greek: to di ssol ve). 1878, K uhne used the In understanding ability of students. good teacher A word enzyme (Creek: in yeast)to indicate the is always a good catalystin students'life! catalysistaking place in the biological systems. Enzymes may be defined as biocatalysts lsolation of enzyme systemfrom cell-free extract synthesized by living cells. They are protein in of yeast was achieved in 1883 by Buchner. He nature (exception - RNA acting as ribozyme), named the active principle as zymase (later colloidal and thermolahile in character, and found to contain a mixture of enzymes),which specific in their action. could convert sugar to alcohol. ln 1926, James of f nzymesare biocatalysts the catalysts life. L A catalvsf is defined as a subsfance that increases ihe velocity or rate of a chemical reactionwithout itselfundergoingany change in the overall process. 85
  • 92. 86 BIOCHEMISTF}Y Sumner first achieved the isolation and crystallizationof the enzyme ureasefrom jack bean and identified it as a protein. In the early days, the enzymes were given names by their discoverers in an arbitrary manner. For example,the namespepsin,trypsin and chymotrypsinconvey no informationabout the function of the enzyme or the nature of the substrateon which they act. Sometimes,the was added to the substrate naming for suffix-ase the enzymese.g. lipaseacts on lipids; nuclease on nucleic acids; lactaseon lactose.These are known as trivial names of the enzymes which, however, fail to give complete information of enzyme reaction (type of reaction, cofactor requirementetc.) undertwo Enzymes sometimes are considered : (a) Intracellular enzymesbroad categories They are functional within cells where they are synthesized.(b) Extracellular enzymes- These enzymes are active outside the cell; all the digestiveenzymesbelong to this group. (l U The Internati onal ni on of B i ochemi stry U B ) i C appoi nted E nzyme ommi ssi onn 1961.Thi s an committeemade a thoroughstudy of the existing enzymesand devised some basic principlesfor of and nomenclature enzymes. the classification Since 1964, the IUB system of enzyme classification has been in force. Enzymes are divided into six major classes(in that order). the Eachclasson its own represents generaltype of reaction brought about by the enzymes of that class(Table 6.1. Enzyme classwith examples* Reactioncatalysed 1. Oxidoreductases (alcohol ------+Reduction Alcohol dehydrogenase : NAD* oxidoreductase E.C. 1.1.1.1.), Oxidation A H r+ B -> A + B H , Lacid cytochrome oxidase,andD-amino oxidases 2. Transferases (ATP 2.7.1.1.), Group transfer Hexokinase : D-hexose E.C. 6-phosphotransferase, phosphorylase A+ A-X+ B------+ B-X transmethylases, transaminases, 3. Hydrolases (triacylglycerol hydrolase 3.1.1.3), Lipase acyl E.C. choline phosphatases, pepsin, esterase, andalkaline acid urease Hydrolysis AH A- B + H"O------+ + BOH 4. Lyases (ketose 1-phosphate lyase, Aldolase aldehyde E.C.4.1.2.7), fumarase, histidase 5. lsomerases phosphale (D-glyceraldehyde Triose isomerase 3-phosphate ketoisomerase, E.C. retinol 5.3.1.1), isomerase, phosphohexose isomerase 6. Ligases (L-glutamate synthetase ammonia ligase, 6.3.1.2), E.C. Glutamine acetyl carboxylase, succinate thiokinase CoA *For oneenzynein eachclass,systenatic n"r, Addition ------+ Elimination A- B+i- v- - Ax- Bi lnterconversion of isomers A-> A' (usually on Condensation dependent ATP) A+B;z-5-+A-B pi ATP ADp+ ttorg *,rh E.C. nunbetis givenin the brackets.
  • 93. Chapter 6 : ENZYMES 1. Oxidoreductases: Enzymes involved in reactions. oxidation-reduction made up of apoenzyme (the protein part) and a coenzyme (non-protein organic part). : 2. Transferases Enzymesthat catalysethe transferof functional groups. Holoenzyme -----+ Apoenzyme + Coenzyme (active (protein part) (non-protein part) enzyme) 3. Hydrolases : Enzymes that bring about hy dr oly s is f v a ri o u sc o m p o u n d s . o The term prosthetic group is used when the non-protein moiety tightly (covalently) binds with the apoenzyme. The coenzyme can be separated dialysisfrom the enzyme while the by prostheticgroup cannot be. 4. Lyases : Enzymes specialised in the additionor removalof water, ammonia,COr etc. : 5. lsomerases Enzymesinvolved isornerization reactions. the the synthetic 6. ligases : Enzymescatalysing :eactions (Creek : ligate-to bind) where two nrolecules are joined togetherand ATP is used. lThe word OTHLIL (first letter in each class) rray be memorisedto rememberthe six classes of enzymes in the correct orderl. E ac h c la s s i n tu rn i s s u b d i v i d e di n to manv which are further divided. A four sub-classes digit E nz y me C o m m i s s i o n (E C .) n u m ber i s the class to assigned each enzyme representing ( f ir s tdigit ) ,s u b -c l a s (s e c o n d i g i t),s u b -s u b ass d cl s ( t hir d digit ) a n d th e i n d i v i d u a l e n z y m e (fourth digit ) . E ac h e n z y m e i s g i v e n a s p e c i fi c name coenzyme (if any) and indicating the substrate, by the type of the reactioncatalysed the enzyme. Although the IUB names for the enzymes are they have not been specific and unambiguous, accepted for general use as they are complex and cumbersometo remember. Therefore,the t r iv ial nam e s , a l o n g w i th th e E.C . n u mbersas and when needed, are commonly used and widely accepted. The word monomeric enzyme is used if it is made up of a si ngl e pol ypepti de e.g. ri bonucl ease, trypsi n. S ome of the enzymesw hi ch possessmore than one polypeptide (subunit) chain are known as oligomeric enzymes e.g. lactate dehydrogenase, aspartate transcarbamoylase etc. There are certain multienzyme complexes possessingspecific sites to catalyse differentreactions a sequence. in Only the native i ntactmul ti enzyme compl exi s functi onal l y acti ve and not the individualunits, if they are separated e.g. pyruvatedehydrogenase, fatty acid synthase, prostaglandin synthase etc. The enzymesexhibit all the generalproperties proteins(Chapter4). of Genetic engineering and modified enzymes Recentadvancesin biotechnologyhave made it possibleto modify the enzymeswith desirable characters-improved catalytic abilities,activities under unusual conditions. This approach is required since enzymes possess enormous potenti alfor thei r use i n medi ci neand i ndustry. Hybrid enzymes : lt is possibleto rearrange genesand produce fusion proteins. e.g. a hybrid enzyme (of gl ucanaseand cel l ul ase)that can more efficiently hydrolyse barley p-glucans in beer manufacture. Site-directed mutagenesis : Th is is a technique used to produce a specifiedmutation position in a DNA molecule. A ll t he e n z y me s a re i n v a ri a b l yp ro tei ns.In at a predetermined recent years, however, a few RNA molecules The result is incorporationof a desired amino have been shown to function as enzvmes.Each acid (of one's choice) in place of the specified and specific ami no aci d i n the enzyme.B y thi s approach,i t enzymehas its own tertiarystructure to conformation which is very essential for its i s possi bl e producean enzymew i th desi rabl e e.g. activator c at aly t ic ac ti v i ty . T h e fu n c ti o n a l u n i t o f the characteristics. tissueplasminogen enzyme is known as holoenzymewhich is often (used to lyse blood clots in myocardial
  • 94. 88 B IOC H E MIS TF|Y 2" Concentration + of substrate Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels.A rectangular hyperbolais obtainedwhen velocity is plotted against the substrate concentration(Fig.6.2). Three distinct phasesof the reactionare observedin the graph (A-linear; B-curve;C-almostunchanged). I I I I o -9 o o E N t! 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. Order of reaction : When the velocity of the reaction is almost proportionalto the substrate (i.e. [S] is lessthan Kn,),the rate of concentration the reaction is said to be first order with respect to substrate.When the ISJ is much greater than Kn', the rate of reaction is independent of substrate concentration, and the reaction is said to be zero order. Enzyme kinetics and K, value : The enzyme (E)and substrate combine with each other to (S) form an unstableenzyme-substrate complex (ES) for the formation of product (P). ln recentyears,it has also become possibleto produce hybrid enzymes by rearrangement of k1. E+ S r, -- rs S r +p genes. Another innovative approach is the ' kproduction of abzymes or catalytic antibodies, Here kl , k2 and k3 represent the velocity the antibody enzymes. constants for the respective reactions, as indicated by arrows. K' the Michaelis-Menten constant(or Brigrs and Haldane's constant),is given by the formula The contact between the enzyme and substrateis the most essentialpre-requisitefor enzyme activity. The important factors that influencethe velocity of the enzyme reactionare discussedhereunder t. Goncentration of enzyme K' = kz + kr k, The following equation is obtained after sui tabl eal gebrai cmani pul ati on. v= Vr"* [S] K m+ [S ] (1 equation ) where v = Measuredvelocity, = Maximum velocitv, Vtu" = Substrate S concentration, = Michaelis Mentenconstant. K. - 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 Let us assume that the measured velocity(v) the serumenzymesfor the diagnosis diseases. of is equalto f Vrr". Thentheequation maybe (1) By using a known volume of serum,and keeping substituted follows as all the other factors (substrate,pH, temperature 1v etc.) at the optimum level, the enzyme could be _ v.axlsl assayedin the laboratory. 2.max K . +[ s ]
  • 95. 89 Ghapter 6 : ENZYMES since V."" is approached asymptotically. By taking the reciprocals of the equation (1), a straightline graphic representation obtained. is Vr"* -J T I I I -F 1V.", g 1_ Km .. 1 V v-"" - --+ Substrate concentration Fig. 6,2 : Effect of substrate concentration on enzyme velocity (A-linear; B-curve; C-almost unchanged). 1-= K m., -,.-i--TiV V..,. r^1 [".| -----------i--T [s] v'"- [sJ ---1- 1 IS.| 1 Vr"* The above equation is similar to y = ax + b. Therefore, plot of the reciprocalof the velocity a concenI ' I ur. the reciprocalof the substrate vi 2vmax [S] Vr"* Km+ [S] - K n ' + [S ] = 2 [S ] Km = [S] K stands for a constant and m stands for M ic ha e l i s(i n Kn .). ! K^ or lhe Michaelis-Menten constant is defined as the substrate concentration (expressed moles/l) to produce half-maximum in velocity in an enzyme catalysed reaction. lt indicatesthat half of the enzyme molecules(i.e. 50%) are bound with the substratemolecules when the substrate concentrationequalsthe K. v alue . / The Lineweaver-Burk plot is shown in Fig.6.3. lt is much easier to calculate the K. from the intercept on x-axis which is -(l/Km). Further,the double reciprocal plot is useful in understanding the effect of various inhibitions (discussed later). Enzyme reactions with two or more substrates: The above discussionis based on the presumption of a single substrate-enzyme reaction. In fact, a majority of the enzymecatalvsed reactions involve two or more substrates. Even in case of multisubstrate K. value is a constant and a characteristic feature of a given enzyme (comparable to a t humb i m p re s s i o n o r s i g n a tu r e). l t i s a represe;;rtative measuringthe strengthof ES for complex. A low K^ value indicates a strong affinity between enzyme and substrate, whereas a high K. value reflects a weak affinity between them. For majority of enzymes,the K. values are in the range of 10-s to 10-2 moles. lt may however,be noted that K. is not dependenton the concentrationof enzvme. 1 _1 Lineweaver-Burk double reciprocal plot : For the determination of K, value, the substrate saturation curve (Fig.5.2) is not very accurate _ tration l--l-- | givesa straightline. Here the slope tsl /is K./y'.u* and whose y intercept is 1/y'."*. Km tsl plot, Ftg,6.3 : Lineweaver-Burk doublereciprocal
  • 96. 90 B IOC H E MIS TRY enzymes, despite the complex mathematical expressions, fundamentalprinciplesconform the to Michaelis-Menten Kinetics. 3. Effect of temperature Velocityof an enzyme reactionincreases with increasein temperatureup to a maximum and then declines. A bell-shaped curve is usually observed (Fig"6.a). Ternperaturecoefficient or Qto is defined as increase in enzyme velocity when the temperature increased 10"C. For a majority is by af enzymes, Qlo is 2 between 0"C and 40oC. lncrease in temperature results in higher activation energy of the molecules and more moiecuiar (enzyme and substrate) collision and interaction for the reaction to oroceed faster. I o q) E N UI The optimum temperature for most of the It is worth noting here that the enzymeshave enzymes is between 40'C-45'C. However, a few been assigned optimal temperatures basedon the enzymes (e.g. venom phosphokinases, muscle laboratory work. These temperatures,however, adenylate kinase) activeeven at 100'C. Some may have less relevance and biological are plant enzymeslike ureasehave optimum activity significancein the living system. around 60'C. This may be due to very stable structureand conformationof these enzymes. 4. Effect of pH In general,when the enzymesare exposedto lncrease in the hydrogen ion concentration a temperature above 50"C, denaturation leading (pH) influences the enzymeactivity considerably to derangementin the native (tertiary)structure a bell-shaped curve is normally obtained and of the protein and active site are seen.Majority (Fig.6.5). Each enzyme has an optimum pH at of the enzymes become inactive at higher w hi ch the vel oci ty i s maxi mum. B el ow and temperature(above 70'C). above this pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive. I Most of the enzymes of higher organisms show optimum activity around neutral pl-l (6-8). Thereare, however,many exceptionslike pepsin (1-2), aci d phosphatase(4-5) and al kaline (10-X 1).E nzyrnes phosphatase from fungi and plants are most active in acidic pH (a-6). o o) o E N Hydrogen ions influencethe enzyme activity on by al teri ngthe i oni c charges the ami no acids (particularly at the active site), substrate,ES complex etc. ttl 20 30 40 50 60 ("C) Temperature Fig" 6.4 : Ef'fect of Iempenture on enzyme velocity. 5, Effect of product concentration The accumulation of generally decreases the reaction products enzyme velocity.
  • 97. Chapter 6 : ENZYMES For certainenzymes,the productscombine with the active site of enzyme and form a loose c om plex a n d , th u s , i n h i b i t th e e n z y me acti vi ty. ln the living system,this type of inhibition is generally prevented by a quick removal of productsformed. The end product inhibition by feedback mechanismis discussedlater. 6" Effect Active site 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,anionsare also needed for enzyme activity e.g. chloride ion (C11 for amylase. Metals function as activators of enz y m ev e l o c i tyth ro u g hv a ri o u sme c h ani smscombining with the substrate, formation of ES-metalcomplex, direct participation in the reaction and bringing a conformationalchange in t he en z y me . Fig. 6.6 : A diagrammatic representation of an enzyme with active site. L Effect of light and raEliation Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivatescertain enzyrnes due to the formation of peroxides.e.g. UV rays i nhi bi t sal i varyamyl aseacti vi ty. Two categories enzymes requiring metals of fbr their activity are distinguished . Metal-activated enzymes : The metal is not tightly held by the enzyme and can be exchangedeasily with other ions e.g. ATPase(Mg2* and Ca2*) Enzymes are big in size compared to substrates which are relativelysmaller.Evidently, a smal l porti on of the huge enzyme mol ecul ei s di rectl y i nvol ved i n the substrate ndi ng and bi caialysis(Fig.6,6). Enolase(Mg2*) The active site (or active centre) of an enzynte represents as the small region at wkich hold " Metalloenzymes : These enzymes the metals rather tightly which are not tke suhstrate(s) binds and participates in the r eadily e x c h a n g e d . e .g .. a l c o h o l d ehydro- aatalysis. genas e ,c a rb o n i c a n h y d ra s e ,a l k a l ine phosphatase, carboxypeptidase and aldolase Salient features of active site c ont ai n z i n c . 1 . The existenceof active site is due to the Phenol oxidase (copper); Pyruvateoxidase (manganese); X ant hi n eo x i d a s e(m o l y b d e n u m ); Cytochromeoxidase (iron and copper). 7. Effect of time Under i d e a l a n d o p ti m a l c o n d i ti o n s(l i ke pH , iemperature etc.), the time required for an enzyme reactionis less.Variationsin the time of the reaction are generally related to the alterationsin pH and temperature. tertiary structure of protein resulting in threedi mensi onal nati ve conformati on. 2. The active site is made up of amino acids (known as catalytic residues) which are far fronr each other i n the l i nearsequence ami no aci ds of (primary structLrre protein). For instance,the of enzyme lysozyme has 129 amino acids. The activesite is formed by the contributionof amino aci d resi dues numbered35, 52, 62, 63 and 101. 3. Active sites are regarded as ctrefts or crevices pocketsoccupying a small region in or a bi g enzyme mol ecul e.
  • 98. B IOC H E MIS TFIY 92 4. The active site is not rigid in structure and shape. lt is rather flexible to promote the specific substrate binding. 5. Cenerally, the active site possessesa substrate binding sife and a catalytic site. The of latter is for the catalysis the specific reaction. 6. The coenzymes or cofactors on which some enzymesdepend are presentas a part of the catalytic site. Enzyme-inhibitor complex 7. The substrate(s) binds at the active site by weak noncovalentbonds. 8. E n z y m e s re s p e c i fi ci n th e i r fu ncti ondue a to the existenceof active sites. 9. T h e c o mmo n l y fo u n d a m i n o a ci ds at the active sites are serine, aspartate, histidine, lysine,arginine,glutamate, tyrosineetc. cysteine, Among these amino acids, serine is the most frequentlyfound. C= Substrate Active site Non-competitive inhibitor Enzyme-inhibitor comprex Fig. 6.7 : A diagrammatic representation of (A) Competitive and (B) Non-competitive inhibition. 10. The substratelslbinds the enzyme (E) at the active siteto form enzyme-substrate complex l. Competitive inhibition : The inhibitor (l) (ES). The product(P) is released afterthe catalysis (S) the real substrate is which closely resembles and t he e n z y m e i s a v a i l a b l efo r re u s e . regarded as a substrateanalogue. The inhibitor and binds at the active competeswith substrate site of the enzyme but does not undergo any catalysis.As long as the competitive inhibitor holds the active site,the enzyme is not available to for the substrate bind. During the reaction,ES and El complexesare formed as shown below Enzyme inhibitor is defined as a substance ES--+E P + whic h bi n d s w i th th e e n z y m e a n d b ri n gsabout a decreasein catalyrtc activity of that enzyme. EI T he inh i b i to r m a y b e o rg a n i c o r i n organi c i n nature. There are three broad categories of of ano The relativeconcentration the substrate enz y m e i n h i b i ti o n inhibitor and their respectiveaffinity with the il:+:1-i.' P 1 . Re v e rs i b l en h i b i ti o n . i 2. I r r e v e rs i b l en h i b i ti o n . i 3. A ll o s te ri ci n h i b i ti o n . ,' 1 T he i n h i b i i o r b i n d s n o n -c o v a l e ntl y w i th enz y m e a n d th e e n z y me i n h i b i ti o n can be reversed if the inhibitor is removed. The r ev er s ib l en h i b i ti o n i s fu rth e r s u b -d i v i dedi nto i l. Rev e rs i b l e i n h i b i ti o n l. Competitiveinhibition (Fig.6.7A) ll. Non-competitiveinhibition (Fig.6.7B) enzyme determinesthe degree of competitive i nhi bi ti on.The i nhi bi ti oncoul d be overcomebv a high substrateconcentration.ln competitive whereas V-u* inhibition, the K- value increases remains unchanged (Fig.6.A. (SDH) The enzyme succinatedehydrogenase i s a cl assi cal exampl eof competi ti vei nhi bi ti on with succinic acid as its substrate. The namel y,mal oni c aci d, gl utari caci d compounds, si and oxal i c aci d, have structural mi l ari tyw i th for succinicacid and competewith the substrate bi ndi ng at the acti ve si te of S D H .
  • 99. 93 chaprer 6 : ENZYMES cooH cH2cooH cH2cooH Succinic acid I CHr t- cooH Malonic acid enzyme surface. Thi s bi ndi ng i mpai rs the enzyme function. The inhibitor has no structural However, there resemblance with the substrate. usuallyexistsa strongaffinity for the inhibitor to bind at the secondsite. In fact, the inhibitor does binding. not interferewith the enzyme-substrate But the catalysisis prevented,possiblydue to a distortion in the enzyme conformation. Methanol is toxic to the body when it is converted to formaldehyde by the enzyme alcohol dehydrogenase (ADH). Ethanol can compete with methanolfor ADH. Thus, ethanol The i nhi bi tor general l y bi nds w i th the can be used in the treatment of methanol enzyme as well as the ES complex. The overall por s o n rn S . rel ati on i n non-competi ti ve i nhi bi ti on is Some more examples of the enzymes with representedbelow and competitive inhibitors(of clinical substrates l ) and p h a rma c o l o g i c as i g n i fi c a n c eare gi ven i n E+sirES-*E+P Table 6.2. ++ I I Antimetabolites : These are the chemical compounds that block the metabolic reactions E I+ S E IS by t h e i r i n h i b i to ry ,a c ti o n ' o n enzymes. are usually structuralanalogues Antimetabolites For non-competitiveinhibition, the K^ value and thus are competitive inhibitors of substrates (Table 6.2). They are in use for cancer therapy, is unchanged while V^^* is lowered (Fig.5.9). Bout etc. The term antivitamins is used for the Heavy metal ions (Ag+, Pb2+,Hg2+ etc.) can w ant im e ta b o l i te s h i c h b l o c k th e bi ochemi cal non-competitively inhibit the enzymes by ac t io n s o f v i ta m i n s c a u s i n g d e fi ci enci es,e.g. binding with cysteinyl sulfhydryl groups. The s ulph o n i l a mi d ed i c u ma ro l . , general reactionfor Hg2+ is shown below. ll. Non-competitiveinhibition : The inhibitor + E -S H + H gt* i ^ E -S . . .H g2+ H + binds at a site other than the active site on the .lf tf I f/ 2'mu + I I Km Km, 11 Km (A) 1 Km' tsl (B) 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).
  • 100. B IOC H E MIS TR Y 94 Enzyme Substrate lnhibitor(s) Allopurinol Significance of inhibitor(s) of to excess in Used thecontrol gout reduce production acid hypoxanthine. of uric from Xanthine oxidase Hypoxanthine xanthine Monoamine oxidase levels. for catecholamine Ephedrine, Useful elevating Catecholamines (epinephdne, norepinephrine) amphetamine Dihvdrofolate reductaseDihvdrofolic acid in of and Aminopterin, Employed thetreatment leukemia cancers. amethopterin, other methotrexate Acetylcholine esterase Acetylcholine relaxation, in in lor cholineUsed surgery muscle Succinyl patients. anaesthetised Dihydropteroate synthase Para aminobenzoic acid (PABA) acid. synthesisfolic of bacterial Sulfonilamide Prevents Vitamin epoxide K K Vitamin Dicumarol Actsas an anticoagulant. Lovastatin, compactin biosynthesis Inhibit cholesterol reductase HMG reductase HMG CoA CoA These i nhi bi tors are usual l v toxr c Heavv metals also lead to the formation of i rreversi bl e. covalent bonds with carboxyl groups and poisonoussubstances. inhibition. histidine, often resultingin irreversible inhibitor of the is lodoacetate an irreversible papain and glyceraldehyde enzymes like inhibition 2. lrreversible lodoacetate combines 3-phosphate dehydrogenase. (-SH) groupsat the activesite of The inhibitors bind covalently with the with sulfhydryl enzymes and inactivate them, which is theseenzvmesand makesthem inactive. 7 1/ 2 vm ax T
  • 101. Ghapter 6 : ENZYMES Diisopropyl fluorophosphafe (DFP) is a nerve uridylatewhich inhibitsthe enzyme thymidylate gas developed by the Cermans during Second synthase, and thus nucleotidesynthesis. World War. DFP irreversibly binds with enzymes containing serine at the active site, e.g. serine 3. A l l osteri c i nhi bi ti on proteases, acetylcholine esterase. The detailsof this type of inhibition are given Many organophosphorus insecticides like under allosteric regulation as a part of the m elat h i o na re to x i c to a n i m a l s(i n c l udi ngman) regulation of enzyme activity in the living as they block the activity of acetylcholine svstem. esterase (essential for nerve conduction), resultingin paralysisof vital body functions. Disulfiram (Antabuse@) a drug used in the is treatment of alcoholism. lt irreversiblvinhibits the enzyme aldehyde dehydrogenase. Alcohol addicts, when treated with disulfiram become sick due to the accumulation of acetaldehyde, leadingto alcohol avoidance.(Nofe ; Alcohol is metabolized by two enzymes. lt is first acted upon by alcohol dehydrogenase to yield acetaldehyde.The enzyme aldehyde dehydrogenaseconvertsacetaldehyde acetic acid.) to T he p e n i c i l l i n a n ti b i o ti c sa c t a s irreversi bl e inhibitors of serine- containing enzymes, and block the bacterialcell wall svnthesis. lrreversibleinhibitors are frequently used to identify amino acid residues the active site of at the enzymes, and also to understand the mechanismof enzyme action. S nic ide i n h i b i ti o n Enzymesare highly specific in their action when comparedwith the chemicalcatalysts. The occurrence of thousands of enzymes in the biological system might 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 : Stereoisomers are the comoounds which have the same mol ecul arformul a, but di ffer i n the ir structuralconfiguration. The enzymes act only on one isomer and, S uic i d e i n h i b i ti o n i s a s p e c i a l i zedform of therefore,exhibit stereospecificity. ir r ev ers i b l en h i b i ti o n .l n th i s c a s e ,the ori gi nal i inhibitor (the structural analogue/competitive e.g. L-amino acid oxidase and D-amino acid inhibitor) is convertedto a more potent form by oxidase act on L- and D-amino acids the sameenzyme that ought to be inhibited.The respectively. so formed inhibitor binds irreversiblywith the Hexokinase acts on D-hexoses; enzyme. This is in contrast to the original Glucokinaseon D-glucose; inhibit o rw h i c h b i n d s re v e rs i b l y . Amylase acts on a-glycosidic linkages; A g o o d e x a m p l e o f s u i c i d e i n hi bi ti on i s allopurinol (used in the treatment of gout, Refer Cellulasecleavesp-glycosidicbonds. Chapter lV. Allopurinol, an inhibitor of Stereospecificity explained by considering is xanthineoxidase,getsconvertedto alloxanthine, three distinct regions of substrate molecule a more effective inhibitor of this enzyme. specifically binding with three complementary The use of certain purine and pyrimidine regionson the surfaceof the enzyme (Fig.5.l0). analog u e si n c a n c e r th e ra p y i s a l s o expl ai ned The class of enzymes belonging to isomerases on t he b a s i s s u i c i d e i n h i b i ti o n . F o r i nstance, do not exhihit stereospecificity, since they are S-fluorouracil gets converted to fluorodeoxy- specializedin the interconversion isomers. of
  • 102. 96 B IOC H E MIS TF| Y glycosidasesacting on glycosidic bonds of carbohydrates, lipasescleavingesterbonds of l i pi ds etc. . Broad specificity : Some enzymes act on closely relatedsubstrates which is commonly known as broad substrate specificity, e.g. hexokinase actson glucose, fructose/mannose and glucosamine and not on galactose. lt i s possi bl e that some structural si mi l arit y among the first four compounds makes them a common substratefor the enzyme hexokinase. The protein part of the enzyme,on its own, is not always adequateto bring about the catalytic activity. Many enzymes require certain nonprotein small additional factors, collectively referred to as cofactors for catalysis. 2. Reaction specificity : The same substrate The cofactors may be organic or inorganic in can undergo different types of reactions, each nature. catalysed by a separate enzyme and this is referredto as reaction specificity. An amino acid The non-protein, organic, Iow molecular can undergo transamination,oxidative deami- weight and dialysable substance associated with nation, decarboxylation,racemizationetc. The enzyme function is known as coenzyme. enzymes however, are different for each of these The functional enzyme is referred to as reactions (For details, refer Chapter |fl. holoenzyme which is made up of a protein part part and a non-protein 3. Substrate specificity : The substrate (apoenzyme) (coenzyme); The term prosthetic group is used specificity variesfrom enzymeto enzyme. lt may when a non-protein moiety is tightly bound to be either absolute,relativeor broad. the enzyme which is not easily separableby . Absolute substrate specificity : Certain dialysis. The term activator is referred to the enzymes act only on one substrate e.g. inorganic cofactor (like Ca2+,Mg2+, Mn2+ etc.; glucokinase acts on glucoseto give glucose6- necessary enhance enzyme activity. lt may, to phosphate,urease cleaves urea to ammonia however, be noted that some authors make no and carbon dioxide. distinction between the terms cofactor, . Relative substrate specificity : Some enzymes coenzyme and prostheticgroup and use them interchangeably. act on structurallyrelatedsubstances. This, in turn, may be dependenton the specificgroup Coenzymes are second substrates : or a bond present.The action of trypsin is Coenzymes are often regarded as the second a good example for group specificity (Refer substrates or co-substrafes, since thev have Fig.8.7). Trypsin hydrolyses peptide linkage affinitywith the enzyme comparablewith that of inv olvi n g a rg i n i n e o r l y s i n e . C h y motrypsi n the substrates. Coenzymes undergo alterations cleaves peptide bonds attached to aromatic during the enzymatic reactions,which are later amino acids (phenylalanine, tyrosine and regenerated.This is in contrast to the substrate tryptophan). Examples of hond specificity- which is convertedto the product. Fig. 6.10 : Diagrammatic representation of stercospecificity (a', b', Cl-three point attachment of .l
  • 103. 97 Chapter 6: ENZYMES Coenzyme (abhreviation) Derived from vitamin (TPP) pyrophosphate Thiamine (FMN) Flavin mononucleotide (FAD) Flavin adenine dinucleolide Thiamine Aldehyde keto or Transketolase Riboflavin Hydrogen electron and L - Amino oxidase acid Atom or group transferred Dependentenzyme (example) Riboflavin D - Amino oxidase acid Niacin Lactate dehydrogenase acid Lipoic Pyridoxine Pantothenic acid Acyl Thiokinase Folic acid ?ieeril Amino keto or Glucose Sphosphate dehydrogenase Pyruvate dehydrogenase complex Alanine transaminase Onecarbon (formyl, methenyl etc.) Formyl transferase CO, Pyruvate carborylase giell . i gqogn c9o!a1 !9!n!c9{a11 o_e gsv_l i1 _ _ 9o$t1ry Methyl/isomerisation Methylmalonyl mutase CoA * Detak each in 7on for coenzyne given Chapter vitanins are Coenzymes participate in various reactions involving transfer of atoms or groups like hydrogen,aldehyde, keto, amino, acyl, methyl, carbon dioxide etc. Coenzymesplay a decisive r ole in e n z y me fu n c ti o n . Table. 6.3, a summary of the vitamin related coenzymes i th thei r functi onsi s gi ven. w Non-vitamin coenzymes: Not all coenzymes are vitamin derivatives.There are some other organic substances, which have no relationwith vitamins but function as coenzymes.They rnay be consi deredas non-vi tami ncoenzymese.g . A TP , C D P , U D P etc. The i mportantnon-vi tamin coenzymesalong with their functions are given in Tahle 6.4. Coenzymesfrom B-complex vitamins : Most of the coenzymes are the derivativesof water soluble B-complex vitamins. In fact, the biochemicalfunctionsof B-complexvitaminsare The coenzymes. exertedthrough their respective Nucleotide coenzymes : Some of the chapteron vitaminsgivesthe detailsof structure nitrogenous base,sugar and and function of the coenzymes(Chapter V. ln coenzymespossess Coenzyme Adenosine triphosphate Abbreviation ATP Biochemical functions phosphate, Donates monophosphate adenosine adenosine and (AMP) moieties. diphosphate Cytidine CDP Uridine diphosphate UDP SAM S- Adenosylmethionine (active methionine) phosphosulfate Phosphoadenosine (active sulfate) in Requiredphospholipid synthesis carrier choline as of and ethanolamine. (glucose, required lor Carrier monosaccharides galactose), of synthesis. $ycogen group biosynthetic Donates methyl in reactions. Donates sulfate thesynthesismucopolysaccharides. for of
  • 104. 9B BIOCHEMISTF|Y phosphate. Such coenzymes are, therefore, regarded as nucleotides e.g. NAD+, NADP+, FMN, FAD, coenzyme A, UDPC etc. Coenzymesdo not decide enzyme specificity : in may participate catalytic A particular coenzyme reactions along with different enzymes. For instance, NAD+ acts as a coenzyme for lactate dehydrogenaseand alcohol dehydrogenase.ln NAD+ is involved both the enzymaticreactions, in hydrogen transfer. The specificity of the enzyme is mostly dependent on the apoenzyme and not on the coenzvme. Catalysisis the prime function of enzymes. The nature of catalysis taking place in the biologic a l s y s te m i s s i mi l a r to th a t of nonFor biologicalcatalysis. any chemical reactionto occur, the reactantshave to be in an activated stateor transitionstate. Enzymes lower activation energy : The to energy required by the reactants undergothe reaction is known as activation energy. The reactants when heated attain the activation energy. The catalyst (or the enzyme in the biological system)reducesthe activationenergy and this causes the reaction to proceed at a lower temperature.Enzymes do not alter the th equilibr i u m c o n s ta n ts , e y o n l y e n hance the velocity of the reaction. The role of catalystor enzyme is comparable wit h a t u n n e l m a d e i n a mo u n ta i nto reducethe barrier as illustrated in Fig.6.l1. The enzyme lowers energy barrier of reactants, thereby making the reaction go faster. The enzymes reduce the activationenergy of the reactantsin occur such a way that all the biological systems at body temperature(below 40"C). Enzyme.substrate complex formation + I I I I lLl B Fig. 6.11 : Effect of enzyme on activation energy af a reaction (A is the substrate and I is the E + S$ E S ---+ E + P A few theorieshave been put 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 Thi E bi ochemi st, mi l Fi scher. s i s i n fact the very first model proposed to explain an enzyme cataiysedreaction. According to this model, the structure or of conformation the enzymeis rigid.The substrate fits to the binding site (now active site)just as a key fits into the proper lock or a hand into the properglove.Thusthe activesiteof an enzymeis template where only a a rigid and pre-shaped can bind. This model does not specificsubstrate give any scopefor the flexible natureof enzymes, hencethe model totallyfailsto explainmany facts the of enzymaticreactions, most importantbeing the effect of allostericmodulators. Induced fit theory The prime requisite for enzyme catalysis is model or Koshland's that the substrate(S) must combine with the Koshland, in 1958, proposed a more enzyme (E) at the active site to form enzymesubstratecomplex (ES)which ultimately results acceptable and realistic model for enzymecomplex formation.As per this model, substrate in the product formation (P).
  • 105. Ghapter 6 : ENZYMES 99 MECHANISIII OF ENZYME GATATYSIS The formation of an enzyme-substrate compl ex (E S )i s very cruci al for the catal ysi s to occur, and for the product formation. lt is estimated that an enzyme catalysed reaction proceeds106 to 1012 ti mes fasterthan a noncatalysedreaction.The enhancementin the rate of the reaction is mainly due to four processes : 1. A ci d-base catal ysi s; 2. Substrate strain; 3. C oval entcatal ysi s; 4. Entropy effects. Fig. 6.12 : Mechanism of enzyme-substrate (ES) complex formdtion (A) Lock and key model (B) Induced fit theory G) Substrate strain theory. The the active site is not rigid and pre-shaped. essential features the substrate binding site are of presentat the nascent active site.The interaction of the substrate with the enzyme inducesa fit or in a conformation changein the enzyme,resulting the formation of a strong substratebinding site. Further, due to inducedfit, the appropriate amino acids of the enzymeare repositioned form the to (Fig.6.12). activesiteand bring aboutthe catalysis t 1 . Acid-base catalysis : Role of acids and basesis quite important in enzymology.At the physi ol ogi cal , hi sti di ne s the most i mportant pH i amino acid, the protonated form of which functi ons as an aci d and i ts correspondi ng conjugateas a base. The other acids are -OH group of tyrosine, -SH group of cysteine,and e-aminogroup of lysine.The conjugates these of acids and carboxyl ions (COO-) function as bases. R i bonucl ease hi ch cl eavesphosphodi ester w bonds i n a pyri mi di nel oci i n R N A i s a cl assi ca l example of the role of acid and base in the catal ysi s. 2. Substratestrain : lnduction of a strain on the substrate ESformationis discussed for above. During the courseof strain induction,the energy level of the substrateis raised, leading to a Induced fit model has sufficientexperimental transitionstate. evidence from the X-ray diffraction studies. K os hlan d ' smo d e l a l s o e x p l a i n s th e a cti on of The mechanismof lysozyme (an enzyme of allostericmodulatorsand competitive inhibition tears,that cleavesp-1,4 glycosidicbonds)action on enzymes. is believed to be due to a combination of substrate strain and acid-basecatalysis. Substrate strain theory 3. Covalent catalysis : In the covalent In this model, the substrate straineddue to catalysis,the negatively is charged (nucleophilic) the inducedconformation changein the enzyme. or posi ti vel y charged (el ectrophi l i c) group i s It is also possiblethat when a substrate binds to present at the active site of the enzyme. This the preformedactive site, the enzyme inducesa group attacks the substrate that results in the strain to the substrate.The strained substrate covalent binding of the substrate the enzyme. to leads to the formation of product. ln the serine proteases(so named due to the ln fact, a combination of the induced fit presence of serine at active site), covalent model with the substrate strain is consideredto catalysisalong with acid-base catalysisoccur, e.g. chymotrypsin,trypsin, thrombin etc. in the enzymatic action. be operative
  • 106. 100 B IOC H E MIS TFIY 4. Entropy effect : Entropy is a term used in t her m o d y n a m i c sl.t i s d e fi n e d a s th e extent of dis or de ri n a s y s te m. h e e n z y m e s ri ng about a T b decreasein the entropy of the reactants.This enables the reactants to come closer to the enzyme and thus increasethe rate of reaction. 3. Endothermic (endergonic) reactions : E nergy i s consumed i n these reacti ons e.g. glucokinase + Glucose6-phosphate ADP Clucose+ ATP------+ I n t h e a c tu a l c a ta l y s i s f th e e n z y mes,more o than one of the processes acid-basecatalysis, substratestrain, covalent catalysisand entropy T ar e s im u l ta n e o u s lo p e ra ti v e . h i s w i l l hel p the y In biological system, regulation of enzyme to substrate(s) attain a transitionstate leadingto activities occurs at different stages in one or the formation of products. more of the fol l ow i ng w ays to achi evecel l ul a r economy. T } I E RM O D YN AMIC S OF 1. A l l osteri cregul ati on ENZYMATIC REACTIOITS 2. Activation of latent enzymes The enzyme catalysed reactions may be 3. Compartmentation of metabolic broadly grouped into three types based on pathways (e n e rg y ) o n s i d e ra ti o ns. t her m od y n a m i c c 4. Control of enzyme synthesis 1 . lsothermic reactions : The energy 5. E nzymedegradati on exchange between reactants and products is g l y c o g e np h o s p h o ry l a se negligib l ee .g . 6. l soenzymes Cly co g e n+ Pi --+ C l u c o s e 1 -p h osphate 2. Exothermic(exergonic)reactions: Energy is liber a te di n th e s e re a c ti o n s .s . u re ase e Urea --+ NH3 + CO2 + energy i. & i l ssteri e regul ati on ;rnsl al l o* teri e i nhl hi ti on additionalsites, Someof the enzymespossess known as allosteric sites (Greek : allo-other), EIOMEDICAL CLINICAL CONCEPTS / s€ The existence lit'e is unimaginablewithout the presenceol enzymes-the biocotalysts. ot' 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 competitiue inhibitors include aminopterin used in the treatment of cancers,sult'anilamideas antiboctericidalogent and dicumarol as on anticoagulant.. P- The nerue gas(diisopropyl t'luorophosphate), t'irst developed by Germans during Second World Wa4 tnhibits acetylcholine esterqse,the enzyme essentialfor nerve conduction and paralyses the uital body functions. Many organophosphorus insecticides (e.9. melathion) also block the actiuity of acetylcholine esterase. te Penicillin antibiotics irreuersiblyinhibit serine contqining enzymesof bacterial cell wall sunthesis.
  • 107. 101 Ghapter 6: ENZYMES change in the active site of the enzyme, leading to the inhibition or activation of the catalytic activity (Fig.6.l3). In the concerted model, allostericenzvmes exist in two conformational states-the T (tenseor taut) and the R (relaxed). The T and R states are i n equi l i bri um. (or) Allosteric activator substrate Allosteric inhibitor Allosteric inhibitors favour T state whereas activators and substratesfavour R state. The substrate can bind only with the R form of the enzyme.The concentration enzvme molecule of in the R state increasesas more substrateis added, therefore the binding of the substrateto besides the active site. Such enzymesare known the allostericenzyme is said to be cooperative. as allosteric enzymes. The allosteric sites are Allosteric give a sigmoidal enzymes curve (instead unique p l a c e so n th e e n z y me mo l e c ul e. of hyperbola) when the velocity (v) versus Allosteric effectors : Certain substances substrate(S) concentrationare plotted (Fig.5.14). referred to as allosteric modulators (effectorsor The term homotropic effect is used if the modifiers)bind at the allostericsite and regulate substrate influences the substrate binding the enzyme activity. The enzyme activity is through allosteric mechanism, their effect is increased when a positive (+) allostericeffector always positive. Heterotropic effecf is used binds at the allosteric site known as activator when an allostericmodulatoreffectsthe binding site. On the other hand, a negative(-) allosteric of substrate to the enzyme. Heterotropic effector binds at the allosteric site called interactions are either positive or negative. inhibit ors i te a n d i n h i b i tsth e e n z y m e acti vi ty. Selected examples of allosteric enzymes Classes allosteric enzymes : Enzymesthat responsible for rapid control of biochemical of are regulated by allosteric mechanism are pathways are given in Table 6.5. referred to as allosteric enzymes. They are divided into two classes basedon the influence of allostericeffectoron K, and V.r*. Fig. 6.13 : Diagrammatic representation an of allastericenzyme(A) T-farm;(B] P-foffn; (C] Effect of aftag"eie ffitW1.(D) Etleotaf altoqtide:ac,.4vaiw, . - l" . K-class of allosteric enzymes, the effector changes the K. and not the V."". Double reciprocal plots, similar to competitive inhibiti o n a re o b ta i n e d e .g . phosphofructokinase. . V-classof allosteric enzymes,the effectoralters the V.r* and not the Kr. Double reciprocal plots resemble that of non-competitive inhibition e.g. acetyl CoA carboxylase. Conformational changes in allosteric enzymes : Most of the allosteric enzymes are oligom eri c i n n a tu re . T h e s u b u n i ts may be identical or different. The non-covalent reversible binding of the effectormolecule at the allosteric site brings about a conformational Hyperbolic 1 o o E N E lrJ Substrate ------+ concentration Fig. 6.14 : Effect of substrate concentration an allos-
  • 108. ElIOCHEMISTFIY l02 Allosteric Activator Enzyme Metabolicpathway Inhibitor Hexokinase Glycolysis 6-phosphate Glucose Phosphofructokinase lsocitrate dehydrogenase Pyruvate carborylase 1, Fructose6 - bisphosphatase phosphate I synthetase Carbamoyl Tryptophan oxygenase Acetyl carboxylase CoA Glycolysis Krebs cycle Gluconeogenesis Gluconeogenesis Urea cycle Tryptophan metabolism Fatty synthesis acid ATP ATP Feedback regillation The process ol inhibiting the first step by the final product, in a series of enzyme catalysed reactions of a metabolic pathway is referred to as feedback regulation. Look at the series of reactionsgiven below A el >B sp ,9 € 9a )C rD rE B, A is the initial substrate, C, and D are the intermediatesand E is the end product, in a pathway catalysed by four different enzymes (e1, e-2, e4).The very first step (A -+ B by the e3, 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 increasedlevels of end product synthesis. This is will result in its (er) decreased a real cellular economy to save the cell from the wasteful expenditure of synthesizing a compound which is alreadyavailablewithin the c ell. Feedbackinhibition or end product inhibition is a specialised type of allosteric inhibition necessaryto control metabolic pathways for efficient cellular function. ADP AMP, NADADP, Acetyl CoA AMP Palmitale N- Acetylglutamate L- Tryptophan lsocitrale Carbamoylphosphate+ Aspartate Feedback control Carbamoyl aspartate + Pi I Y (CTP) Cytidinetriphosphate Carbamoyl phosphateundergoesa sequence of reactionsfor synthesisof the end product, CTP. When CTP accumulates,it allosterically transcarbamoylase inhibitsthe enzyme aspartate by a feedback mechanism. Feedback regulation or feedback inhibition? a Sometimes distinction is made between these two usages.Feedback regulation representsa phenomenonwhile feedbackinhibition involves the mechanism of regulation. Thus, in a true sense,they are not synonymous.For instance, hepaticcholesterol dietary cholesteroldecreases biosynthesisthrough feedback regulation.This does not i nvol ve feedback i nhi bi ti on, si nce dietary cholesteroldoes not directly inhibit the regulatory enzyme HMG CoA reductase. However, the activity of gene encoding this by enzyme is reduced (repression) cholesterol. Aspartate transcarbamoylase (ATCase) is of latent enzymes a good example of an allosteric enzyme 2. Activation Latent enzymes,as such, are inactive.Some inhibited by a feedback mechanism. ATCase catalysesthe very first reaction in pyrimidine enzymes are synthesized as Proenzymes or zymogens which undergo irreversiblecovalent biosynthesis.
  • 109. 103 Ghapter 6 : ENZYMES There are some enzymeswhich are active in activation by the breakdown of one or more peptidebonds.For instance, proenzymes -namely dephosphorylatedstate and become inactive pepsinogen chymotrypsinogen, and plasminogen, when phosphorylatede.g. glycogen synthase, - convertedto the active enzymes acetyl CoA carboxylase. are respectively pepsin and plasmin. chymotrypsin, A few enzymesare activeonly with sulfhydryl Certain enzvmes exist in the active and (-SH) groups, €.8. succinate dehydrogenase, inactive forms which are interconvertible, urease.Substances like glutathionebring about depending on the needs of the body. The the stabilityof these enzymes. interconversion is brought about by the E -S H + E -S H reversible covalent modifications, namely E-S-S-E Reduced phosphorylation and dephosphorylation, and Oxidised GS-SG inactive 2G-SH active oxidation and reduction of disulfide bonds. Clycogen phosphorylase a muscle enzyme is that breaks dow'n glycogen to provide energy. This enzyme is a homodimer (two identical subunits) forms. and existsin two interconvertible Phosphorylase (dephospho b enzyme)is inactive which is convertedby phosphorylation serine of residuesto active form phosphorylase The a. inactiveenzyme phosphorylase is producedon b dephosphorylation illustratedbelow. as E Phosphorylase b (inactive). .,-_- ' P .-....-. phosphatase /' E 2Pi Organelle ______----l P Phosphorylase a z (active) _./ 3. Gompartnnentation Thereare certainsubstances the body (e.g., in fatty acids,glycogen)which are synthesized and also degraded. Thereis no point for simultaneous occurrenceof both the pathways.Cenerally,the synthetic (anabolic) and hreakdown (catabolic) pathways are operative in different cellular organelles achieve maximum economy. For to 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 hormonal and other controlsfatty acids are either synthesized oxidized. or The intracellularlocation of certain enzymes and metabolic pathways is given in Table 6.6. En zym e/metaboIi c pathway Cytoplasm peptidases; glycolysis; monophosphate fatty Aminotransferases; hexose shunt; acid purine pyrimidine catabolism. synthesis; and Mitochondria Fatty oxidation; acid acid amino oxidation; cycle; synthesis; Krebs urea electron phosphorylation. transport and chain oxidative Nucleus Biosynthesis and ofDNA RNA. (microsomes) phospholipid Endoplasmic reticulum Protein biosynthesis;triacylglyceroland synthesis; synthesis steroid and P4Eo; reduction; cytochrome esterase. Lysosomes phosphatases; phospholipases; proteases; nucleases. Lysozyme; hydrolases; lipases; Golgiapparatus glucosyF galactosyl-transferases. 5'-nucleotidase; and Glucose 6-phosphatase; Peroxisomes D-amino oxidase; chain acid urate acid long fatty oxidation. Catalase; oxidase;
  • 110. I l04 4" Control I I B IOC H E MIS TR Y of enzyme synthesis Most of the enzymes, particularly the rate limiting ones, are present in very low the concentration.Nevertheless, amount of the enzyme directly controls the velocity of the reaction, catalysed by that enzyme. Many rate Iimiting enzymes have short half-lives. This helps in the efficient regulationof the enzyme levels. There are two types of enzymei-(a) Constitutive enzymes (house-keeping enzymes)-the levels of which are not controlled and remain fairly constant. (b) Adaptive enzymes-their concentrations increase decrease per body or as needs and are well-regulated.The synthesisof enzymes (proteinsl is regulated by the genes (Refer Chapter 25). ! { I ln general,the key and regulatoryenzymes are most rapidly degraded.lf not needed, they i mmedi atel y di sappear and, as and w hen Though required,they are quickly sysnthesized. not always true, an enzyme with long half-life is usually sluggishin its catalytic activity. 6. lsoenzymes Multiple forms of the same enzyme will also help in the regulationof enzymeactivity, Many of the isoenzymes are tissue-specific.Although isoenzymesof a given enzyme catalysethe same reaction, they differ in K' V.nu* or both. e.g. of isoenzvmes LDH and CPK. Induction and repression The term induction : is used to represent increased synthesis of enzyme while repressionindicates its decreased in Enzymes are never expressed termsof their synthesis. Induction or repression which (as mg or pg etc.), but are ultimatelydetermines the enzyme concentration concentration gene level through the mediation of expressedonly as activities. Various methods at the have been introduced for the estimation of hormonesor other substances. enzyme activities (particularly for the plasma Examples enzyme induction : The hormone of enzymes). In fact, the activities have been insulin induces the synthesis of glycogen expressedin many ways, like King-Armstrong synthetase, glucokinase, phosphofructokinase units, Somogyi units, Reitman-Frankelunits, and pyruvate kinase. All these enzymes are units etc. spectrophotometric inv olv e d i n th e u ti l i z a ti o n o f g l ucose. The hormone cortisol inducesthe synthesis many of Katal enzymes e.B. pyruvate carboxylase, tryptophan oxygenaseand tyrosine aminotransferase. In order to mai ntai n uni formi ty i n th e (as units) Examplesof repression: In many instances, expression of enzyme activities substrate can repress the synthesis of enzyme. worldover, the EnzymeCommissionof IUB has radical changes.A new unit- namely Pyruvate carboxylaseis a key enzyme in the suggested as glucose from non-carbohydrate katal (abbreviated kat)-was introduced.One synthesis of pyruvateand amino acids.lf there is kat denotes the conversion of one mole sourceslike per second(mol/sec).Activity may also sufficientglucoseavailable,there is no necessity substrate (mkat), microkatals for its synthesis. This is achieved through be expressedas millikatals (pkat) and so on. repression of pyruvate carboxylase hy glucose. 5. Enzyme degradation Enzymes are not immortal,since it will create a seriesof problems.There is a lot of variability in the half-lives individualenzymes.For some, of it is in days while for others in hours or in m inut e s ,e .g . L D H a - 5 to 6 d a y s ; L D H I - 8 to 12 hou rs ;a m v l a s e-3 to 5 h o u rs . International Units (lUf Some workers preferto use standardunits or One Sl unit or Sl units (SystemInternational). (lU) is defined as the amount InternationalUnit of enzyme activity that catalysesthe conversion of one micromol of suhstrate per minute. Sl units and katal are interconvertible.
  • 111. Chapter 6 : ENZYMES = 60 pkatal (or) 1 nkatal = 1 .6 7 l U 1lU Enzyme Laboratory use of enzyme units In the clinical laboratories, however, the units- namelv katal or Sl units-are vet to find a place. Many investigators use the old units still like King-Armstrongunits, Somogyi units etc. while expressingthe enzyme activities. lt is therefore, essential that the units of enzyme activity, along with the normal values, be invariably stated while expressing the enzymes for comparison. Ribozymes Ribozymes are a group ol ribonucleic acids that function as biological catalysts, and they are regardedas non-proteinenzymes. Application Therapeutic applications Streptokinase/urokinaseToremove clots blood Asparaginase Incancer therapy Paoain Anti-inf lammatory o,-Antitrypsin Totreat emphysema (breathing ditficulty due todistension oflungs) Analytical application reagents(for estimation) Glucose oxidase oeroxidase and Glucose Urease Cholesterol oxidase Uricase Lipase Luciferase Urea Cholesterol Uric acid Triacylglycerols Todetect bacterial contamination offoods Alkalinephosphatase/ Intheanalyticaltechnique lgpp-n9irl eereriq?ee _qL!94 Applications genetic in engineering A lt ma n a n d h i s c o w o rk e rs ,i n 1 983, found Restriction endonucleases Gene lransfer, tinger DNA that ribonuclease an enzyme till then known Ppnnrng to cleave precursors tRNAs to give tRNAsof polymerase Iag DNA Polymerase chain was functional due to RNA component present reaction in the enzyme and not the protein part of the enzyme. The RNA part isolated from ribonucleaseP exhibiteda true enzyme activityand also obeyed Michaelis-Menten kinetics. Later studies have proved that RNA, in fact, can function as an enzyme and bring about the catalysis. RNA moleculesare known to adapt a tertiary structure just as in the case of proteins (i.e. enzymes). The specific conformation of RNA for may be responsible its function as biocatalyst. It is believed that ribozymes IRNAs) were functioningas catalysts beforethe occurrenceof protein enzymesduring evolution. Industrialapplications preparation Cheese Production ofhigh fructose syrup Infood industry to convert starch glucose to powder Washing Rennin Glucose isomerase u,-Amylase Proteases Enzymes as therapeqtic agents prepared 1. Streptokinase from streptococcus is useful for clearing the blood clots. plasmaplasminogen Streptokinase activates to plasmin which, in turn, attacks fibrin to convert into solubleproducts. Plasminogen I Certain enzymes are useful as therapeutic genetic agents, analytical reagents, in m anip u l a ti o n sa n d fo r i n d u s tri a l a ppl i cati ons (Table 6.V. Streptokinase J Plasmin I -l-* Fibrin (clot) products Soluble
  • 112. B IOC H E MIS TR Y 106 2. The enzyme asparaginaseis used in the Tumorcellsare dependent of treatment leukemias. on asparagineof the host's plasma for their the asparaginase, By multiplication. administering Estimationof enzyme activities in biological are host'splasmalevelsof asparagine drastically fluids (particularly plasma/serum)is of great in reduced.This leadsto depression the viability E cl i ni cal i mportance. nzymesi n the ci rcul ati on of t um o r c e l l s . are divided into two Eroups plasma functional pl asmanon-functi onal . and reagents Enzyrmes as analytica! Some enzymes are useful in the clinical laboratory for 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 catalytic agents in industrial and medical applications. Some of theseenzymesare immobilized by binding-.them t o a s o l i d , i n s o l u b l e ma tri x w h i c h w i l l not affect the enzyme stability or its catalytic activity. Beaded gels and cyanogen bromide activated sepharose are commonly used for . o im m ob i l i z a ti o n f e n z y m e s T h e b o u nd enzymes for can be preserved long periodswithout lossof activity. l. Fl asma speei fi c or P l astna functional enzYrnes Certain enzymesare normally presentin the plasma and they have specific functions to perform. Cenerally, these enzyme activitiesare higher in plasma than in the tissues.They are mostly synthesizedin the liver and enter the ci rcul ati on e.g. l i poprotei n l i pase, pl asmi n , ceruloplasminetc. thrombin, choline esterase, l mpai rment i n l i ver functi on or genet ic disordersoften leadsto a fall in the activitiesof plasma functional enzymes e.g' deficiency of i cerul opl asmi nn W i l son' s di sease. 2, i{on-piasrma specific or plasrna enzymes non-functional 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, lipase etc.) present in the plasma are known as secretory immobilized enzymes. All the other plasma enzymes Clucoseoxidaseand peroxidase, and coated on a strip of paper, are used in the associated with metabolism of the cell are for clinical laboratory the detectionof glucosein collectivefy referred to as consfitutive enzymes u r ine. acid transaminases, (e.g. lactatedehydrogenase, creatine phosphophosphatases, and alkaline xidgg9 t Gluconic kinase). acid+ Hro, Glucose Hzoz o-Toluidine (colourless) Hzo toluidine Oxidized (bluecolour) Estimation of the activities of non-plasma specific enzymes is very important for the diagnosisand prognosisof severaldiseases. The normal serum level of an enzyme and indicatesthe balance between its synthesis on releasein the routine cell turnover. The raised The intensityof the blue colour depends the concentrationof glucose. Hence, the strip enzyme l evel scoul d be due to cel l ul ardamag e, estimation increasedrate of cell turnover, proliferationof method is usefulfor semi-quantitative of synthesis enzymesetc. Serum cells, increased of gluc o s ei n u ri n e .
  • 113. Chapten 6 : ENZYMES Serum enzyme (elevated) 107 (most important) Disease Amylase glutamate pyruvate (SGPT) Serum transaminase (hepatitis) Liver diseases glutamate (SGOT) Serum oxaloacetale transaminase (myocardial Heart attacks infarction) phosphatase Alkaline jaundice Rickets, obstructive phosphatase Acid gland Cancer prostate of (LDH) Lactate dehydrogenase Heart attacks, diseases liver phosphokinase (CPK) Creatine Myocardial (early infarction marker) Aldolase Muscular dystrophy 5'-Nucleolidase Hepatitis yGlutamyl (GGT) transpeptidase l', pancreatitis Acule Alcoholism enzymes are conveniently used as markers to It may be noted that SCPTis more specificfor det ect th e c e l l u l a r d a m a g e w h i c h ul ti matel y the di agnosi s l i ver di seases hi l e S C OT i s for of w helps in the diagnosis of diseases. heart diseases. This is mainly becauseof their cel l ul ar di stri buti on S C P T i s a cytosom al A summary of the important enzymes useful enzyme while SCOT is found in cytosol and f or t he d i a g n o s i s f s p e c i fi cd i s e a sess gi ven i n o i mi tochondri a. Table6.8. Detailedinformationon the diagnostic enzymes including referencevalues is provided Alkaline phosphatase (ALP) : lt is elevated in in Table 5.9. A brief account of selected certain bone and liver diseases (normal 3-13 KA diagnosticenzymes is discussed units/dl). ALP is useful for the diagnosis of rickets, hyperparathyroidism, carcinoma of Amylase : The activity of serum amylase is bone, and obstructive jaundice. increasedin acute pancreatitis (normal 80-180 Somogyi units/dl).The peak value is observed Acid phosphatase(ACP) : lt is increased in ,ithin 8-12 hours after the onset of disease the cancer of prostate gland (normal 0.5-4 KA rvhich returns to normal by 3rd or 4th day. units/dl). The tartarate labile ACP (normal<1 KA Elevated activityof amylaseis also found in urine units/dl)is usefulfor the diagnosis and pi-ognosis of the patients of acute pancreatitis. Serum of prostate cancers i.e. ACP is a good tumor anrylaseis also important for the diagnosisof marker. chronic pancreatitis, acute parotitis(mumps)and Lactatedehydrogenase(LDH): LDH is useful obstructionof pancreaticduct. for the diagnosis of myocardial infarction, Alanine transaminase(ALT/SGPT): SCPT is infective hepatitis, leukemia and muscular elevated in acute hepatitis of viral or toxic dystrophy(serumLDH normal 50-200 lull). LDH or igin ,j a u n d i c ea n d c i rrh o s i s f l i v e r (normal3- has five isoenzymes,the details of which are o J,,r lUll). describedlater. Aspartate transaminase (AST/SGOT) : SCOT activity in serum is increased in myocardial iniarction and also in liver diseases(normal - 1- . + it u l l ). Creatine kinase (CK) . lt is elevated in myocardial infarction (early detection) and muscul ardystrophy(normal10-50 l U l l ). C K has (describedlater). three isoenzymes
  • 114. o o Enzymes l. Dig,estive enzYmes Amylase Lipase ll. Transaminases (ALT) Alanine transaminase or serum glutamate pyruvate (SGPT) transaminase (AST) Aspartate transaminase or glutamate serum oxaloacetate (SGOT) transaminase lll. Phosphatases phosphatase (ALP) Alkaline (pHoptimum 9-10) (ACP) Acidphosphatase (pHoptimum #S) lV. Enrymes carbohydrate of metabolism Aldolase (lCD) lsocitrate dehydrogenase (LDH) Lactate dehydrogenase V. Miscellaneous enzynes (GK)or creatine kinase Creatine phosphokinase (CPK) Reference value Disease(s) which increased in pKat 80-180 Somogyi units/dl 2.5-5.5 or 0.2-1.5lu/l pancreatitis, (acute parotitis), in duct, Acule mumps obstructionpancreatic severe diabetic ketoacidosis. pancreatitis, Acute moderate elevationcarcinomapancreas. in of nKat 3-40lu/l or 40-250 jaundice, (viralor toxic), Acutehepatitis cirrhosis liver. of nKat 4-45lu/l or 5G-320 Myocardial infarction, diseases, cancer, liver liver cirrhosis liver. ot (related higher (KA) activity)-rickets, Pagets'disease, hyperparaIn adults-$13 Armstrong units/dl Bone King diseases to osteoblastic nKat. thyroidism, ol or 2$-90lU/lor 500-1400 carcinomabone. jaundice infective of Inchildren-l Klr/dl Liver diseases-obstructive (cholestasis), hepatitis, cirrhosis liver. 5-30 gland IU/' Prostatic i.e. of liabile seryes a marker as 0.5-4KAuniWdl 2.5-12 or carcinoma cancer prostate (tartarate ACP nKat. labile fordiagnosis follow Pagets' and up), disease. or 10-100 Tanarate ACP-G{.9 units/dl KA 2-6 tu/l 1-4 tu/l lu/l 50-200 or 1-5 pKat gravis, liver myocardial infarction, myasthenia leukemias Muscular dystrophy, diseases, (inflammatory or malignant) toxic Liver diseases pemicious hepatitis, muscular leukemia, Myocardial infarction, intective acute dystrophy, anaemE. 1150tu/l (CK Myocardial infarction useful early for detection), muscular dystrophy, hypothyroidism, alcoholism. Nephrotic syndrome, myocardial infarction jaundice, Hepatitis, obstructive tumors jaundice. infective hepatitis, obstructive Alcoholism, pregnancy. Bacterial infections, collagen diseases, cirriosis, (ChEl) Cholinesterase 2-10ru/l phosphatase 2-15 lu/l (NTP) S'-Nucleotidase or nucleotide (GGT) yGlutamyl transpeptidase 5-40 tu/l (tenooxidase) Ceruloplasmin 2tr50mg/dl E o o I m 6 n
  • 115. Chapter 6 : ENZYMES Reference values Disease(s) which decreased in Amylase (ChE Pseudocholinesterase ll) 8G-1 Somogyi 80 units/dlLiver diseases 10-20 tu/dl Viral hepatitis, malnutrition,cancer, liver cirrhosisliver of Ceruloplasmin 20-50 mg/dl Wilson's disease (hepatolenticular degeneration) (G6PD) Glucose 6-phosphate dehydrogenase inRBC 121260 tU/1012 RBC Congenital deficiency hemolytic with anemia y-Glutamyl transpeptidase (GGT) : lt is a sensitivediagnosticmarker for the detection of alcoholism. GGT is also increased in infective hepatitisand obstructivejaundice. Decreased plasma enzyme aetivities 3. An enzyme may be active as monomer or oligomer e.g. glutamatedehydrogenase. 4. In glycoprotein enzymes, differences in carbohydrate content may be responsiblefor isoenzymes e.g. alkaline phosphatase. Sometimes, the plasma activities of the lsoenzymes of lactate enzymesmay be lower than normal which could dehydrogenase (LDHI be due to decreased enzyme synthesis or Among the isoenzymes,LDH has been the congenital deficiency. ln Table 5.10, the most thoroughly investigated. decreasedplasma enzymes in certain disorders are given. LDH whose systematic name is L-lactateNAD+ oxidoreductase(E.C. 1 .'1.1.27)catalyses the interconversion of lactate and pyruvate as shown below The multiple forms of an enzyme catalysing LDfI ? cH3-cH-@oH CH the same reaction are isoenzymes or isozymes. 3-e-C OOH They, however, differ in their physical and oH NAD+NADH H+ + chemical properties which include the structure, Lactic acld Pyruvicacld -,----+ electrophoreticand immunological properties, LD H has fi ve di sti nct i soenzymesLD H t, K,n and V.nr" values, pH optimum, relative LDH2, LDH3, LDHa and LDH5. They can be susceptibility to inhibitors .and degree of (celluloseor starch separated electrophoresis by denaturation. gel or agarose gel). LDHI has more positive charge and fastest in electrophoreticmobility Explanation for the w hi l e LD H 5 i s the sl ow est. existence of isoenzymes Strdcture of LDH isoenzymes : LDH is an Many possiblereasonsare offeredto explain enzyme made up of four presence isoenzymes the living systems. oligomeric (tetrameric) the of in polypeptide subunits. Two types of subunits . 1 . lsoenzymes synthesized from different namely M (for muscle) and H (for heart) are genes e.g. malate dehydrogenase cytosol is of produced by differentgenes.M-subunit is basic differentfrom that found in mitochondria. w hi l e H subuni t i s, aci di c. The i soenzymes 2. O lig o m e ri c e n z y me s c o n s i s ti n gof more contain either one or both the subunits giving than one type of subunitse.g. lactatedehydro- LDHI to LDH'. The characteristicfeatures of genaseand creatine phosphokinase. LDH isoenzymes are given in Table 5.11.
  • 116. B IOC H E MIS TR Y 110 Isoenzyme Subunit constitution H Principal tissueof origin Electrophoretic mobility Fastest Percentage of Whether normal serum destroyed by heat (at 60"C) in humans No 25To No 35Yo Fast Partially 27To Liver skeletal and muscle Slow Yes 8To muscle liver and Skeletal Slowest Yes 5o/o LDHr Ha LDHz HsM Heart RBC and LDH3 HzMz Brain kidney and LDHr HMs LDHs Ma ffi Heart RBC and @ (c) Fig. 6.15 : Electrophoresis of 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) Significanceof differential catalytic activity : LD H l (H a) i s predomi nantl yfound i n heart muscle and is inhibited by pyruvate- the substrate. Hence, pyruvate is not converted to lactate in cardiac muscle but is converted to acetyl CoA which enterscitric acid cycle. LDH5 (M+) is mostly presentin skeletalmuscle and the i i nhi bi ti onof thi s enzymeby pyruvate s mi ni mal , hence pyruvate is convertedto lactate.Further, w LD H 5 has l ow K . (hi ghaffi ni ty) hi l e LD H l has high Km (low affinity) lor pyruvate. The of catalyticactivities LDHl and LDH5 differential in heart and skeletal muscle, respectively,are of well suited for the aerobic (presence oxygen) of and anaerobic(absence oxygen) conditions, prevai l i ngi n theseti ssues. Diagnosticimportance of LDH : lsoenzymes of val of LD H have i mmense ue i n the di agnosi s heart and Iiver related disorders (Fi9.6.1fl. ln heal thy i ndi vi dual s, the acti vi ty of LD H 2 i s hi gherthan that of LD H l i n serum.In the caseof myocardial infarction, LDHI is much greater than LD H 2 and thi s happensw i thi n 12 to 24 hours after infarction. Increasedactivity of LDH<
  • 117. ENZYMES 111 in serum is an indicator of liver diseases. LDH ac t iv it y i n th e R B C i s 8 0 -1 0 0 ti m e s more than t hat in t h e s e ru m .H e n c e fo r e s ti ma ti o n LD H of or it s is o e n z y me ss e ru m s h o u l d b e to tal l y free , f r om hem o l y s i s r e l s e fa l s ep o s i ti v eresul ts i l l o w be obt ai n e d . In heal thy i ndi vi dual s, the i soenzyme C P K 2 (MB ) i s al most undetectabl ei n serum with less than 2'h of total CpK. After the myocardi ali nfarcti on(Ml ), w i thi rr the fi rst 6_.1 g hours,C P K 2i ncreasesn the serumto as hi eh as i 209l o(agai nst 2oh normal ).C pK ) i soenzvnre ,s not el evated i n skel etal mui cl e di sorders. Therefore, estimationof the enzyme CpKz (MB) is the earliest reliable indication of mvoiardial (C Cr eati n e i n a s e K )o r c re a ti n e h o s phoki nase infarctian. k p ( CP K ) at a l y s e th e i n te r-c o n v e rs i o n p hosphoc s of c r eat ine( o r c re a ti n ep h o s p h a teto c re a ti ne. ) A s many as si x i soenzymesof al kal i ne phosphatase LP )have been i denti fi ed. Lp i s (A A a monomer, the i soenzymes are due to CP K e x i s ts a s th re e i s o e n z y m es. E ach the difference in the carbohydrate content is oenz y me rs a d i me r composed of two (si al i c aci d resi dues). The most i mportanr s ubunit s -M (mu s c l e ) r B (b ra i n )o r b oth. A LP i soenzymesare cx1 Lp, u2-heat l abi l e o -A A LP , o,2-heatstabl e A Lp, pre-B A Lp, y-A Lp Isoenzyme Subunit Tissue origin of etc. cPKl BB Bra i n Increase i n cr2-heat l abi l e A Lp suggests cPK2 MB Heart hepati ti s w hereas pre p-A Lp i ndi cates l tone cPK3 MM S k e l e ta l muscl e d iseases. Phosphocreatin"+ -9{--------lCreatine ADP ATP BIOMEDICAL CHHIGAL CSNCEPTS / 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 specialized form 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 cellular economysince a compound is synthesized onlg when required. r"-; Certain RNA molecules(ribozymes) function as non-protein enzymes.It is belieuedthat ribozgmes were lunctioning as biocatalystsbefore the orrurr"r4 of protein enzymes during euolution. r'>' Certain enzymesare utilized os therapeutic agents. Streptokinosein used to dissolue blood clots in circulation while asparaginose emploged in the treatment is of leukemias. r':' Determination of serum enzyme actiuities is of great importance t'or the diagnosisof seueral diseoses(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 surgery or transportotion of organs lor transplantation surgery.
  • 118. lt2 BIOCHEMISTF|Y Creatine phosphokinase (precisefy isoenzyme MB) is the first enzyme to be released into 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. i o E N ul 0 6 12f8243036 424A*6066724 Hours 5 6 7 I 9 10 11 Days 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 isoenzyme is Americans and Europeans,cx,p1 predominantwhereas in Japanese and Chinese (Orientals) The isomerop2 is mostly present. oB2 more rapidly convertsalcohol to acetaldehyde. Aspartate transaminase(AST or SCOT) rises sharplyafter CPK, and reachesa peak within 48 hours of the myocardial infarction. AST takes 4-5 days to return to normal level. (LDHl) generally rises Lactatedehydrogenase from the second day after infarction, attains a peak by the 3rd or 4th day and takes about 10-15 days to reach normal level. Thus, LDH is the fast enzyme to rise and also the last enzyme to return to normal level in Ml. Cardiac troponins (CT) : Although not enzymes/ the proteins cardiac troponins are hi ghl y useful for the earl y di agnosi sof M l. Among these, troponin I (inhibitory element of actomysin ATPase) and troponin f (fropomysin binding element)are important.Cardiactroponin Accumulation of acetaldehvdeis associated (CTl) | is releasedinto circulation within four with tachycardia (increase in heart rate) and hours after the onset of Ml, reachesa peak value facial flushing among Orientals which is not by 12-24 hours,and remainselevatedfor about commonlv seen in whites. lt is believed that a week. and Chinesehave increased sensitivity Japanese The protein myoglobin is also an early marker to alcohol due to the presenceof ap2-isoenzyme for the diagnosisof Ml. Myoglobin is however, of A D H . 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 For the right diagnosisof a particular disease, The following enzymes-when elevated in it is always better to estimate a few (three or more) serum enzymes, instead of a single serum-are useful for the diagnosis of liver enzyme. Examples of enzyme patterns in dysfunction due to viral hepatitis (jaundice), toxic hepatitis,cirrhosisand hepatic necrosis important diseases are given here. Hnrymes in nnyoeardial infarction 1 . A l ani ne transami nase; 2. Aspartate transaminase; The enzymes namelycreatinephosphokinase 3. Lactatedehydrogenase; (CPK), aspartatetransaminase(AST) and lactate dehydrogenase(LDH)-are important for the The enzymes that markedly increase in diagnosis of myocardial infarction (Ml). The are : intrahepaticand extrahepatic cholestasis elevationof theseenzvmesin serum in relation to '1 Alkaline phosphatase, 2. 5'-Nucleotidase . hours/daysof Ml is given in the Fig.6.l6.
  • 119. 113 Chapter 6 : ENZYMES Diagnostic marker Time of peak elevation Time of return to normal level Diagnostic importance 4-O hrs hrs 20-25 Earliest marker, however cardiac not specific. I Cardiac trooonin 12-24hrs 5-9days Early marker cardiac and specific. troponin T Cardiac 18-36 hrs days 5-14 Relatively marker cardiac early and specific. However, in elevatedother degenerative diseases. phosphokinase (MB) Creatine 20-30 hrs hrs 24-48 Cardiac specific early and marker. (LDH Laclate dehydrogenase l) 48-72 hrs 10-15 days Relatively marker cardiac late and specific. Asparlate transaminase hrs 3048 4-6days Not cardiac specific, is transpeptidase useful in Serum-y-glutamyl the diagnosisof alcoholic liver diseases. (tartaratelabile) is specific for the detection of prostaticcarcinoma. lNote : Prostate specific antigen (PSA; mol wL 32 KD), though not an enzyme, is a more probably to reliable marker for the detection of prostate In themuscular dystrophies, due the increased leakage of enzymes from the cancer. Normal serum concentrationof PSA is damaged cells, serum levels of certain muscle 1-4 n{mll. enzymes are increased.These include creatine A non-specific increase in certain enzymes phosphokinase, aldolase and aspartate like LDH, alkalinephosphatase transaminase and transaminase. these,CPK is the most reliable Of with malignancy in any part indicator of muscular diseases,followed by may be associated of the body. aldolas e . Enzymes in muscle diseases p-C l ucuroni dase mati on n uri ne i s usefu l esti i for detecting the cancers of urinary bladder, Increase in the serum acid phosphatase pancreasetc. Enzymes in cancers
  • 120. 114 B IOC H E MIS TFI Y r' Enzymesare the protein biocatarystssynthesized by the '!ii,iir cers.They are classified c/osse's---'ox uctaies,tran eri idored sf s"r, nvirZ nu, tyases, isomerases 'iuing r, r!ri'"r 2' An enzgm" o to::,r!r-:in its action,possessingactiue site, where the substrate binds form enzgme-substrqte to complex, i"f;;. the product is t'ormed. 3. Factorslike cont xtJ,.!J{j,i{T,zT tr;ti,,:";":i",:! riii:{ii%Ti;'i!"Ji,i;:::{:;:::::;:i, n reuersibte (competitiue, non-competitiue), and ,1,i":,I'f*f."iX'J'"i,,J,i2,,:"Y:::!,bv S Many enzymesrequire prerenceot' non-protein substances .the called cofactors (coenzyntes) Most of tt''' i*r"v-o-Lre"iriwtiues for of B-comptex- uitamins(e.g. NAD+, 'i;;:::7" FAD, 6 The mechanism "I,:rrr",a.ctjo2 is e.xproined recentty induced madet Koshtand) bv tock and keymoder (oJFischer),mare (of fit ,"i ,"triiiJililn ,n.orr. rateof reaction through acid-base catatysis, ' [l"o;:':ff:r7:;;::,'n" couarent catarysis t ,;,!l;i;:,if"i1n]lf,*, thereis a constant regutation enzyme of teuers brought aboutbv sm,actiuation proenzgmes, ot' synthesis degrad"ri"i and etc. .t "*i^Z 9. Estimation of sert serum o*,u;*-;;,:f ,:,,'J",::;i: i,;:::T:: !:':,,,;"!,:*i;i,f:::;:,?1.'f:",:1j,,:::::; hepatitis; aspartate,. trarraminas., lorror" dihgdrogenase"(tDH) phosphokinase(CpK) and creatine i,i.rorrrror,, alkaline phosphatase ^ ^uoroi,"ot in rickets and o,,ia i,n:::i::i::;::;:f inolin'io''.-"'nprostatic ,o,,,no-[; transpepvstutamv! L0' Isoenzymesare the,,murtip,re fotrys of an enzyme catarysingthe same howeue4 dift'er in their phvsicai'rri reoctian which ii"^,car propertie". iD; hasfive isoenzgmes has three' roui""a ciiz;;;'r",u i:i::ri:: important thedragnosis in ot'myocardiar
  • 121. 115 Chapter 6 : ENZYMES I. Essayquestiosrs and their classification nomenclature. Describe 1. What are enzymes? enzymeactiviti. 2. Write an accountof the variousfactorsaffecting of the mechanism enzymeaction. 3. Describe in Write brieflyon the role of coenzymes enzymeaction. 4. What are coenzymes? of of 5. Write an accountof the importance serumenzymesin the diagnosis diseases. lL Short notes (a) Enzyme specificity,(b) Competitive inhibition, (c) Coenzymes,(d) Allosteric enzymes/ (h) (f) (e)lsoenzymes, K, value,(g)Serum in infarction, Lactate dehydrogenase, enzymes myocardial (i) Role of metalsin enzymeaction,(j) Active site. lI L F ill i n th e b l a n k s o 1. T h e l i te ra lme a n i n g f e n z y m ei s reactions are 2. The classof enzymesinvolvedin synthetic part of holoenzyme 3. The non-protein above 70oCdue to 4. Enzymeslose the catalytic activity at temperature zinc are containing of 5. Examples two enzymes and binds with the enzyme 6. The place at which substrate requires the coenzyme dehydrogenase 7. The enzymeglucose6-phosphate rs 8. The E.C.numberfor alcohol dehydrogenase activated by is allosterically 9. Phsophofructokinase infarction 10. The very first enzymeelevatedin serumin myocardial I V . M u l ti p l e c h o i c e q u e s ti o n s namely 11. Pepsinis an examplefor the classof enzymes (d) (c) (b) (a) Oxidoreductases Transferases Hydrolases LiSases. transfer 12. The coenzymenot involvedin hydrogen (a) FMN (b) FAD (c) NADP+(d) FH4. the 13. In the feedbackregulation, end productbinds at (a) Active site (b) Allosteric site (c) E-Scomplex(d) None of these. in activityin serumis elevated 14. y-Clutamyltranspeptidase (d) (c) (a) Pancreatitis Musculardystrophy Myocardialinfarction Alcoholism. (b) 15. In recent years/a non-proteincompound has been identifiedto bring about catalysisin The name of the compoundis biologicalsystem. (a) DNA (b) RNA (c) Lipids(d) Carbohydrates.
  • 122. Vitamins t, ++ Fat Water soluble soluble I t i s d i ffi c u l t to d e fi n e v i ta m i ns preci sel y. 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 does not requireany vitamin, as it can synthesize all of them. lt is believedthat during the course of e v o l u ti o n ,th e a b i l i ty to s y n th esi ze tami ns vi was lost. Hence, the higher organismshave to obt a i nth e m fro m d i e t. T h e v i ta m i nsare requi red in s ma l l a m o u n ts , s i n c e th e i r degradati oni s relativelyslow. an the naturalfoods.Funk (1913)i sol ated act ive pri nci pl e (an ami ne) from ri ce pol i shi ngsand, l ater i n veast, w hi ch coul d cure beri -be r i in pigeons. He coined the term vitamine (Creek : vita-life) to the accessoryfactors with a belief lt that all of them were amines. was later realised that only few of them are amines. The term vitamin, however, is continued without the final letter 'e'. The usage of A, B and C to vitamins was i ntroduced i n 1915 by McC ol l um and Davis. They first felt there were only two vitaminsfat soluble A and water soluble I (anti-beriberi factor). Soon another water soluble anti-scurvy His to ry a n d n o m e n c l a tu re factor named vitamin C was described.Vitamin I n th e b e g i n n i n g o f 2 0 th c e ntury, i t w as A was later found to possess two componentsc lea rl y u n d e rs to o d th a t th e d i ets contai ni ng one that prevents (vitaminA) and night blindness purified carbohydrate, protein, fat and minerals anotheranti-ricketfactor named as vitamin D. A were not adequateto maintain the growth and fat sol ubl efactorcal l edvi tami nE , i n the abs ence health of experimentalrats, which the natural of which rats failed to reproduceproperly, was foods (such as milk) could do. discovered. Yet another fat soluble vitamin Hopkins coined the term accessoryfactors to concerned with coagulationwas discovered in the unknown and essentialnutrientspresent in mi d 1930s. l t w as named as vi tami n K . In t he
  • 123. Chapter 7 : VITAMINS 777 sequenceof alphabets it should have been F, but K was preferred to reflect its function (koagulation). As regardsthe water soluble factors,vitamin C was identifiedas a pure substance and named as ascorbic acid. Vitamin B was found to be a complex mixtureand nomenclature also became complex. B1 was.clearly identified as anti-beriberi factor. Many investigators carried out intensiveresearchbetween 192O and 1930 and went on n a m i n g th e m a s th e w a te r sol ubl e v it am ins82 , 8 3 , 8 4 ,8 5 ,8 6 , 8 7 , 8 6 , B g ,8 19, 811 and 812. Some of them were found to be mixturesof alreadyknown vitamins.And for this reason, a few members (numbers!)of the Bcomplex series disappeared from the scene. Exceptfor 81, Bz, Bo and 812, names are more commonly used for other B-complexvitamins. hematopoietic (folic acid and 812). Most of the water soluble vitamins exert the functions through their respectivecoenzymeswhile only one fat solublevitamin (K) has been identifiedto function as a coenzyme. $ynthesis of vitannims by intestina! bacteria Vitamins, as per the definition, are not synthesized the body. However, the bacteria in of the gut can produce some of the vitamins, required by man and animals. The bacteria mainly live and synthesize vitamins in 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 humansare concerned,it is believed that the normal intestinal bacterialsyntfiesig and of vitamins absorption of vitamin K and biotin may be There are about 15 vitamins, essentialfor sufficient to meet the body requirements. For humans. They are classifiedas fat soluble (A, D, other B-complex vitamins, the synthesis and E and K) and water soluble (C and B-group) absorptionare relatively less.Administrationof vitamins as shown in the Table 7.1. The anitibiotics often kills the vitamin synthesizing B - c om plex v i ta m i n s ma y b e s u b -d i v i d ed i nto bacteria present in the gut, hence additional energy-releasing (81, 82, 86, biotin etc.) and consumptionof vitamins is recommended. Glassification Vitamin A Vitamin D V1amin E Vitamin K I Vitamin C I (Bn) acid l-Folic L-Vitamin 8', (cyanocobalamin) --.._
  • 124. 118 Fa{ soluble B IOC H E MI STRY 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 associated with fat. Thev are soluble in fats and oils and also the fat solvents (alcohol, acetoneetc.). Fat soluble vitaminscan be stored in liver and adiposetissue.They are not readily excreted in urine. Excess consumptionof these vitamins (particularlyA and D) leads to their accumulationand toxic effects. ' Vitamers The term vitamers representsthe chemically similar substances that possess qualitatively similar vitamin activity. Some good examples of vitamersare given below . Retinol,retinal and retinoic acid are vitamers of vitamin A. . Pyridoxine, pyridoxal and pyridoxamine are vitamersof vitamin B.. Alf the fat soluble vitamins are isoprenoid compounds, since they are made up of one or more of five carbon units namely isoprene units ( -C H = C .C H 3 -C H = C H -). F a r sol ubl evi tami ns In the fol l ow i ng pages, the i n dividual perform diverse functions. Vitamin K has a members of the fat soluble and water soluble specific coenzymefunction. vitamins are discussed with regard to the chemistry,biochemicalfunctions,recommended Water sCIluble vitamlels*seneral dietary/dailyallowances(RDA), dietary sources, etc. The water soluble vitamins are a deficiency manifestations heterogenous group 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 readily excreted in 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 vitamins form coenzymes (Refer Table 5.3) that participate in a variety of biochemical reactions,related to either energy generationor hematopoiesis. may be due to lt this reasonthat the deficiencyof vitaminsresults in a number of overlapping symptoms. The common symptomsof the deficiency of one or more vitamins involved in energy metabolism in c l u d e d e rma ti ti s ,g l o s s i ti s(r ed and sw ol l en tongue),cheilitis (ruptureat the cornersof lips), di a rrh e a , m e n ta l c o n fu s i o n , depressi on and malaise. The fat soluble vitamin A, as such is present only in foods of animal origin. However, its provitamins carotenes are found in plants. It is recorded in the history that Hippocrates (about 500 B .C .) cured ni ght bl i ndness. He prescribedto the patients ox liver (in honey), which is now known to contain high quantity of vi tami n A . Chennistry In the recent years, the term vitamin A is collectively used 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 (vitamin A alcohol) : lt is a primary alcohol containingp-ionone ring. The side chain has two isoprenoid units, four double bonds and D e fi c i e n c y f v i ta m i n s 1 , 8 6 and B 12i s more one hydroxylgroup. R eti noli s present n anim al o 8 i closely associated neu with rological manifestations. tissues retinylesterwith long chain fatty acids. as
  • 125. 119 Ghapter 7 : VITAMINS Retinal -C=O I H Retinolc acid -C=O I OH p-lonone (Redcolour reptesents Fig. 7.1 : Structures vitamin and relatedcompounds of A groupsin the respective compounds). the substituent 2. Retinal (vitamin A aldehyde) : This is an aldehyde form obtained by the oxidation of retinol. Retinal and retinol are interconvertible. Previously, the name retinine was used for r et inal. As and when needed, vitamin A is released from the liver as free retinol. lt is believed that 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 association with 3. Retinoic acid (vitamin A acid) : This is pre-albumin.The retinol-RBP complex binds to produced by the oxidation of retinal. However, specific receptors on the cell membrane of retinoic acid cannot give rise to the formationof peripheral tissue and enters the cells. Many retinal or retinol. cells of target tissues contain a cellular retinol4. p-Carotene (provitamin A) : This is found binding protein that carries retinol to the in plant foods. lt is cleaved in the intestineto nucl eus and bi nds to the chromati n (D N A ). produce two moles of retinal. ln humans, this It is here that retinol exerts its function in conversion is inefficient, hence p-carotene a manner analogous to that of a steroid possesses about one-sixth vitamin A activity hormone. compared to that of retinol. Absorption, transport and mobilization BIOCHEMICAL FUNCTIONS Vitamin A is necessary for a variety of functions such as vision, proper growth and Dietary retinyl esters are hydrolysed by differentiatiory reprbductionand maintenance of pancreaticor intestinalbrush border hydrolases epithelial cells. In recent years, each form of in the intestine,releasingretinol and free fatty vitamin A has been assignedspecific functions acids. Carotenes are hydrolysed by p-carotene (Fig.7.3). l5-1S'-dioxygenaseof intestinal cells to release Vitamin A and vision : The biochemicalfunc2 moles of retinalwhich is reducedto retinol. In 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).
  • 126. BIOCHEMISTFIY 120 lntestinalcell p-Carotene j Retinal J Retina All-fransretinol I I + All-transretinal I I J Visual cycle (SeeFig.7.a) Chylomicrons RBP-t Retinol-RBP Nuclear receptor i j v Specificproteins I I + Cell difierentiation functions and transport biochemical A of Fig.7.2 : summary vitamin absorption, bindingprotein)' (FFA-Free faw acid; RBP-Retinol
  • 127. 121 Chapter 7 : VITAMINS Dark adaptationtime : When a person shifts from a bri ght l i ght to a di m l i ght (e.9.entry i nto a dim cine theatre), rhodopsin stores are + depleted and vision is impaired. However, Retinol (steroid hormone--{roMh and difterentiation) within a few minutes,known as dark adaptation time, rhodopsin is resynthesized and vision is improved. Dark adaptationtime is increased in RetinylPhosphate Retinal (visualcycle) (glycoproteinsynthesis; vitamin A deficient individuals. B-Carotene (antioxidant) I I + Retinoicacid (steroidhormone-growth differentiation) and Flg.7.3 : Summary thefunctions of A of vitamin compounds. Rods a n d c o n e s Bleaching of rhodopsin: When exposed to light, the colour of rhodopsinchangesfrom red to yellow, by a process known as bleaching. B l eachi ng occurs i n a few mi l l i secondsand many unstable intermediates formed during are this orocess. Rhodopsin -----) Prelumirhodopsin Lumirhodopsin two types of The retina of the eye possesses All-trans-retinal+ MetarhodopsinMetartdopsin ll +-Opsin +I cells-rods and cones.The human eve has about 10 m illi o n ro d s a n d 5 m i l l i o n c o n e s.The rods Visual cascade and cGMP : When light strikes are in the peripherywhile conesare at the centre the reti na, a number of bi ochemi cal change s of retina. Rods are involved in dim light vision l eadi ng to membrane hyperpol ari zati on occur whereas cones are responsiblefor bright light resul ti ngi n the genesi sof nerve i mpul se.The and c ol o u r v i s i o n . An i m a l s -s u c h a s ow l s and hyperpolarization the membrane is brought of cats for which night vision is more importantabout by a vi sualcascade nvol vi ngcycl i c C MP . i possess mostly rods. When a photon (from ligh$ is absorbed by rhodopsin, metarhodopsinll is produced. The W ald' s v i s u a l c y c l e protein transducin is activated by metarhodopsin an of Rhodopsin(mol. wt. 35,000) is a conjugated l l . Thi s i nvol ves exchange C TP for C D P on proteinpresent rods.lt contains11-crsretinaland inactive transducin. The activated transducin in This the proteinopsin.The aldehydegroup (of retinal)is activates cyclic GMP phosphodiesterase. linkedto e-aminogroupof lysine(of opsin). T he p ri ma ry e v e n t i n v i s u a l cycl e, on of exposureto light, is the isomerization 11-cis: retinal to all-trans retinal. This leads to a c onf or ma ti o n a l c h a n g e i n o p s i n whi ch i s responsible the generationof nerve impulse. for The all-trans-retinal immediately isomerized is (o by r et in a l i s o me ra s e f re ti n a l e p i thel i um) .to 1 1- c is - re ti n a l .T h i s c o mb i n e s w i th opsi n.:to regeneraterhodopsin and complete the visual cycle (Fig,7.4).However, the conversionof all frans-retinalto 1 1-crs retinal is incomplete. Therefore, most of the all-frans-retinal is transported the liver and convertedto all-frans to The all-transretinol by alcbhol dehydrogenase. to retinol undergoesisomerization 1 1-crsretinol whic h i s th e n o x i d i z e d to 1 1 -c i s reti nal to par t ic ip a te n th e v i s u a l c y c l e . i Light (photon) Nerve impulse lsomerase I (' --" 11-cl s-reti nal ' -.--- | All- i,'ansretinal -------------+ 11-cts-retinol +- l1ferf+All-trane.retinol Flg.7.4: Wald's ulsual cycle.
  • 128. 122 B IOC H E MIS TRY synthesis and thus are involved in the cell growth and differentiation. Rhodopsin lu'no'o" to 2. V i tami n A i s essenti al mai ntai nhe alt hv epi thel i al ti ssue. Thi s i s due to the fact t hat retinol and retinoic acid are requiredto prevent (responsible horny surface). for keratin synthesis Metarhodopsin ll from retinol 3. Retinylphosphate synthesized is necessary for the synthesis of certain glycoproteinq which are required for growth and mucus secretion. Phosphodiesterase (inactive) 3',5'-cGMP 51GMP Fig.7.5 : The visual cascade involving cyclic guanosine monophosphate(3" 5' -cGMP). enzyme degradescyclic CMP ir;he rod cells (Fi9.7.5). rapid decreasein cyclic GMP closes A the Na+ channels in the membranesof the rod c el l s . T h i s re s u l tsi n h y p e rp o l a r i zati on hi ch i s w an excitatory responsetransmittedthrough the neuron network to the visual cortex of the brain. 4. R eti noland reti noi c aci d are i nvol v ed in the synthesisof transferrin,the iron transport protein. for to 5. Vitamin A is considered be essential the maintenanceof proper immune system to fight againstvarious infections. 6. Cholesterolsynthesisrequiresvitamin A. Mevalonate,an intermediatein the cholesterol biosynthesis,is diverted for the synthesis of coenzyme Q i n vi tami n A defi ci ency. lt is pertinentto note that the discoveryof coenzyme Q w as ori gi nal l y made i n vi tami n A deficient ani mal s. 7. Carotenor'ds(most important p-carotene) function as antioxidants and reduce the risk of C o n e s a re s p e c i a l i z e di n b ri ght and col our cancers initiated by free radicals and strong vision. Visual cycle comparableto that present is oxidants.p-Carotene found to be beneficialto in ro d s i s a l s o s e e n i n c b n e s .T h e col our vi si on prevent heart attacks.This is also attributedto is governed by colour sensitive pigmentsthe antioxidantproperty. (red), iodopsin (green) and porphyropsin cyanopsin (hlue). All these pigments are retinaldietary Recommended opsin complexes.When bright light strikesthe (RDA) allowance retina, one or more of these pigments are The dai l y requi rement of vi tami n A is ble a c h e d , e p e n d i n g n th e p a rt i cul ar our of d o col expressedas retinol equivalents(RE)rather than light. The pigmentsdissociate all-trans-retinal to (l U ). U and o p s i n ,a s i n th e c a s eo f rh o d opsi n. nd thi s Internati onal ni ts A reactionpasses a nerve impulseto brain as a on 1 retinol equivalent=1 lrg retinol specific colour-red when porphyropsinsplits, =6 Pg P-carotene greenwhen iodopsinsplitsor blue for cyanopsin. = 12 pg othercarote noids Splitting of these three pigments in different = 3.33 l U of vi tamin A proportionsresultsin the perceptionof different activity from retinol colours by the brain. Golour vision Other biochemical functions of vitamin = ' 10 l U of vi tamin A activityfrom p-carotene A '1 Retinol and retinoic acid function almost . like steroid hormones.They regulatethe protein The RDA of vitamin A for adults is arouno (3,500 lU) for man and equivalents !"Q0Oretiytol retinol equivalents(2,500 lU) for woman. 800
  • 129. Chapter 7 : VITAMINS 123 Effect on reproduction : The reproA)tiu" l O ne I nt e rn a ti o n aU n i t (l U ) e q u a l sto 0 .3 mg of i r et inol. T h e re q u i re me n ti n c re a s e s n grow i ng system is adversely affected in vitamin A women and lactatingmothers. deficiency.Degeneration germinalepithelium of children,pregnant leads to sterility in males. Dietary sources Animal sourcescontain (preformed)vitamin A. The best sourcesare liver, kidney, egg yolk, milk, cheese,butter.Fish (cod or shark)liver oils are very rich in vitamin A. Effect on skin and epithelial cells : The skin becomes rough and dry. Keratinization of epithelial cells of gastrointestinal tract, urinary tract and respiratory tract is noticed. This leadsto increased bacterial infection. VitaminA deficiencv is associated with formationof urinary stones. Vegetable sources contain the provitamin The plasma level of retinol binding protein is A-carotenes. Yellow and dark green vegetables decreasedin vitamin A deficiency. and fruits are good sources of carotenese.g. pumpkins,mango, carrots,spinach,amaranthus, Hypervitaminosis A papaya etc. Excessive consumptionof vitamin A leadsto toxicity. The symptoms of hypervitaminosis A Vitamin A deficiency include dermatitis(drying and rednessof skin), The deficiencysymptomsof vitamin A are not enlargement of liver, skeletal decalcification, immediate,sincethe hepaticstores can meet the tenderness of long bones, loss of weight, body requirements for quite sometime (2-4 irritability,loss of hair, joint pains etc. months). The deficiency manifestations are Total serum vitamin A level (normal 20-50 relatedto the eyes, skin and growth. pgldl) is elevated in hypervitaminosis Free A. retinol or retinol bound to plasmalipoproteinsis Deficiency manifestationsof the eyes : Nrghf blindness (nyctalopia) is one of the earliest actually harmful to the body. lt is now believed symptoms of vitamin A deficiency. The that the vitamin A toxicosis symptoms appear indiv idu a l sh a v e d i ffi c u l ty to s e e i n d i m l i ght only after retinol binding capacity of retinol since the dark adaptation time is increased. binding protein exceeds. Prolonged deficiency irreversibly damages a Higher concentrationof retinol increases the num ber o f v i s u a l c e l l s . of The manifes, synthesis lysosomalhydrolases. tations of hypervitaminosis are attributed to A Severe deficiency of vitamin A leads to particularly the destructive action of hydrolases, xerophthalmia. This is characterized by dryness on the cell membranes. of in conjunctivaand cornea,and keratinization epithelial cells. In certain areasof conjunctiva, Beneficial effects of 0.carotene white triangularplaquesknown as Bitot's spots Increased consumption of p-carotene is are seen. associatedwith decreased incidence of heart lf xerophthalmiapersisitsfor a long time, attacks,skin and lung cancers.This is attributed occur. This to the antioxidant role of p-carotenewhich is corneal ulcerationand degeneration of resultsin the destruction cornea,a condition independent its role as a precursorof vitamin of referred to as keratomalacia, causing total A. Ingestionof high doses of p-carotenefor long VitaminA deficiency blindness mostly periods are not toxic like vitamin A. is blindness. countries. common in childrenof the developing Other deficiency manifestations V i tami n D i s a fat sol ubl e vi tami n. l t Effect on growth : Vitamin A deficiency sterolsin structureand functions like resultsin growth retardationdue to impairment resembles a hormone. in skeletalformation.
  • 130. 124 B IOC H E MIS TRY Absorption, tlansport and storage Vitamin D is absorbedin the small intestine for w hi ch bi l e i s essenti al .Through l ym ph, bound to pl asm a vi tami n D enters ci rcul ati on the a2-gl obul i n and i s di stri butedthroughout t he storesmall amounts bodv. Liverand other tissues of vi tami n D . ME TA B OLIS M A N D FU N C TION S B IOC H E MIC A L V i tami ns D 2 and D 3, as such, are not biologically active. They are metabolized identically in the body and convefted to active forms of vi tami n D . The metabol i sm a nd functi ons vi tami n D are depi ct ed of bi ochemi cal in Fig.7.8. (Dr) Ergocalclferol Ftg.7.6 : Formation ergocalciferol ol fromergosterol. The symptoms of rickets and the benefical effects of sunlight to prevent rickets have been known for centuries.Hess (1924) reportedthat ir r ad i a ti o nw i th u l tra v i o l e t l i g h t i nduced anti rachitic activity in some foods. Vitamin D was is ola te d y An g u s(1 9 3 1 ) h o n a m e di t cal ci ferol . b w Ghemistry Synthesis of 1,25-DHCC Cholecalciferolis first hydroxylatedat 25th (25-OH position to 25-hydroxycholecalciferol o3) bv a specific hydroxylasepresent in liver. 25-OH D3 is the major storageand circulatory a form of vitamin D. Kidney possesses specific (calciol) enzyme, 25-hydroxycholecalciferol l -hydroxylase which hydroxylates 25-hydroxycholecalciferolat position 1 to produce 1,25(1,2|-DHCC). 1,25 dihydroxycholecalciferol groups(1,3 and 25 D H C C contai ns3 hydroxyl carbon) hence referred to as calcitriol. Both the hydroxylase enzymes (of liver and kidney) requirecytochromePa56, NADPH and molecular oxygen for the hydroxylation process. The synthesis of calcitriol is depicted in Figs.7,7 and 7.8. Ergocalciferol(vitamin D2) is formed from ergosterol and is present in plants (Fi9.7.6). (v Chol e c a l c i fe ro l i ta m i nD 3 ) i s fo u n d i n ani mal s. Both the sterolsare similar in structureexcept that ergocalciferol has an additional methyl group and a double bond. Ergocalciferol and cholecalciferol are sourcesfor vitamin D activitv and are referred to as provitamins. Regulation of the synthesis of 1125.-DHCC During the course of cholesterolbiosynthesis (Chapter l4), 7-dehydrocholesterol is formed The concentration 1,25-DHCC is regulated of as an intermediate.On exposure to sunlight, by plasma levels of calcium and phosphate. 7-dehydrocholesterolis converted to choleThey control hydroxylationreaction at position c alc i fe ro l i n th e s k i n (d e rm i s a nd epi dermi s) 1. Low plasma phosphateincreases the activity (Fig.2.V Vitamin D is regarded as sun-shine Low iferol 1-hydroxylase. of 25-hydroxycholecalc vitamin. pl asma cal ci um enhances the producti on of The synthesisof vitamin D3 in the skin is parathyroid hormone which in turn activates proportional to the exposure to sunlight. Dark 1-hydroxylase. Thus the action of phosphateis skin pigment lm-qianjn)-adversly influences the di rectw hi l e that of cal ci um i s i ndi recton ki d nev s y nth e s i s ' oc h o l e c a l c i fe ro l . f 1-hydroxyl ase.
  • 131. 125 Ghapter 7 : VITAMINS Biochemical functions Calcitriol (1,25-DHCC) the biologically is active (ormof vitaminD.lt tegulates plasma the Ievels calciumand phospha,fe. of Calcitriolacts (intestine, at 3 different levels kidneyand bonel (normal plasma to maintain calcium 9-11 mg/dl). 7-Ilehydrocholestercl (animals) Sunlight 4S k i n 1. Action of calcitriol on the intestine: Calcitriof increlses the intestinal absorptionof calciumand phosphate. the intestinal In cells, calcitriol receptor form bindswith a cytosolic to a calcitriol-receptor complex. Thiscomplex then approaches nucleusand interacts the with a specific DNA leading to the synthesis a of specific calcium binding protein.This protein increases calciumuptakeby the intestine. the The mechanism action of calcitriolon the of (intestine) similarto the actionof target tissue is a steroidhormone. 2. Action of calcitriol on the bone : In the osteoblasts bone,calcitriol of stimulates calcium phosphate, for uptake deposition calcium as Thus calcitriol essential boneformation. bone is for The is an important reservoir calcium phosphate. of and Calcitriol along with parathyroidhormone increases the mobilizationof calcium and phosphate the bone. from Thiscauses in elevation the plasnla calcium levels. and phosphate 25-Hydroxycholecalciferol (Calcidiol) ICalcidiolo-hydroxylase 1 I | (kidney) J 1,25-Dlhydrorycholecalclferol (1,25DHCC calcitrlol) or 3. Action of calcitriol on the kidney : Calcitriolis also involvedin minimizing the excretion calciumand phosphate of through the kidney, by decreasing their excretion'and enhancing reabsorption. The sequence eventsthat take place in of response low plasmacalciumconcentration to and the actionof calcitriol intestine, on kidney and bone, ultimatelyleadingto the increase in plasma calcium is given in Fi9.7.9. 24,25-Dihydroxychol cal ife rol (24,25 e c -DHCC is anothermetabolite vitamin D, lt is also of synthesized the kidney 24-hydroxylase. in by The function 24,2S-DHCC not known.lt is exact of is believedthat when' calcitriolconcentration is ade