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Enzymology

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For Students in MBBS,BDS,MPT,MSc,
in simple language.

Dr.Kuldip Singh Sodhi
Professor Bio-Chemistry,
MMIMS&R MULLANA

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Enzymology

  1. 1. DEAR STUDENTS ANSWER“ WE ARE THE CATALYSTS OFTHE LIVING WORLD,INCREASE THE RATE OFREACTION THOUSANDTIMES.WORK IN AQUOUSMEDIUM. PROTEIN INNATURE. AND IN ACTIONSPECIFIC, ACCURATE; BIG INSIZE BUT WITH SMALLACTIVE SITE; HIGHLYEXPLOITED FOR DISEASE
  2. 2. ENZYMESDR.K.S.SODHI,M. D. PROFESSORBIO-CHEMISTRYMMIMS&R MULLANA AMBALA.© 2007 Paul Billiet ODWS
  3. 3. The Chemicals of Living Cells ©The Wellcome Trust
  4. 4. HISTORY Of EnzYmESAs early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified.
  5. 5. ENZYMOLOGYContribution of Scientists.Definitions.Mode of Action of Enzymes.Factors Influencing Enzyme Activity.Enzyme Inhibition.Regulation of Enzymes.Diagnostic Importance of Enzymes.Therapeutic Use of Enzymes.
  6. 6. DEFINITIONSHOLOENZYMES ( APOENZYMES+COENZ.)APOENZYMES; SINGLEPOLYPEPTIDECHAIN,MORE THAN ONECHAIN,MULTI-ENZYME COMPLEX.Co-ENZYMES : Non Protein (VITAMINS)METAL-ACTIVATED ENZYMES.(Zn,Cu,Fe,Mg,K,Ca etc.)ZYMASE: Active without modificationZYMOGENS : Pro Enzymes eg.Trypsinogen
  7. 7.  ISO-ENZYMES : Physically distinct perform same function. RIBOZYMES: Small ribonuclear particles. ENDOENZYMES : Produced in the cell. Function inside the cell. EXOENZYMES : Produced inside the cell. Act outside the cell.
  8. 8.  METALLO ENZYMES : Contain metal ions as essential component. HOUSE KEEPING ENZYMES : Levels of Enzymes can not be controlled. Always present in cell. ADAPTIVE ENZYMES : Regulated by genes. Conc.increase or Decrease. KEY ENZYMES :Regulatory eg HMG-CO.A HYBRID ENZYMES :Produced by genetic fusion.
  9. 9.  An additional non- protein molecule COFACTORS that is needed by some enzymes to help the reaction Tightly bound cofactors are called prosthetic groups Cofactors that are bound and released easily are called coenzymes Nitrogenase enzyme with Fe, Mo and ADP Many vitamins are cofactors coenzymes
  10. 10. A GOOD TEACHERIS ALWAYS A GOOD CATALYST IN STUDENTS LIFE.
  11. 11. DISTRIBUTION OF 17 HORSESOLDMAN AND THREE SONS.DISTRIBUTION OF HORSES.ELDER ½MIDDLE 1/3LITTLE 1/9
  12. 12. Notice that without the enzyme it takes a lot more energy for the reaction to occur. By lowering the activation energy you speed up the reaction.
  13. 13. Energy In Reactions Energy is released or absorbed whenever chemical bonds are formed or broken. Because chemical reactions involve breaking and forming of bonds, they involve changes in energy.
  14. 14. Enzymes as Biological Catalysts Enzymes are proteins that increase the rate of reaction by lowering the energy of activation They catalyze nearly all the chemical reactions taking place in the cells of the body Enzymes have unique three- dimensional shapes that fit the shapes of reactants (substrates)
  15. 15. Thermodynamics
  16. 16.   The energies of various stages of a chemical reaction. Substrates need a large amount of energy to reach a transition state , which then decays into products. The enzyme stabilizes the transition state, reducing the energy needed to form products. As all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly.
  17. 17. For example, carbonic anhydrasecatalyzes its reaction in either directiondepending on the concentration of itsreactants. (in tissues ; high CO concentration) 2 in lungs; low CO concentration). 2
  18. 18. Kinetics Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products.The enzyme (E) binds a substrate (S) and produces a product (P).
  19. 19. In 1902 Victor Henri contributewas to think of enzyme reactions in twostages. In the first, the substrate bindsreversibly to the enzyme, forming theenzyme-substrate complex. This is sometimes called theMichaelis complex. The enzyme then catalyzes thechemical step in the reaction and releasesthe product.
  20. 20. In 1902 Victor Henri contributewas to think of enzyme reactions in twostages. In the first, the substrate bindsreversibly to the enzyme, forming theenzyme-substrate complex. This is sometimes called theMichaelis complex. The enzyme then catalyzes thechemical step in the reaction and releasesthe product.
  21. 21. Saturation curve for an enzymereaction showing the relation between thesubstrate concentration (S) and rate ( v).
  22. 22. Enzyme rates depend on solution conditionsand substrate concentration. Conditions that denature the protein abolishenzyme activity, such as high temperatures,extremes of pH or high salt concentrations. while raising substrate concentration tendsto increase activity. Saturation happensbecause, as substrate concentration increases,more and more of the free enzyme is convertedinto the substrate-bound ES form.
  23. 23. At the maximum velocity (Vmax) of theenzyme, all the enzyme active sites arebound to substrate, and the amount of EScomplex is the same as the total amountof enzyme. However, Vmax is only onekinetic constant of enzymes. K m , : is the substrate concentrationrequired for an enzyme to reach one-halfits maximum velocity. Each enzyme has acharacteristic Km for a given substrate. k : is the number of substrate
  24. 24. So The efficiency of an enzyme = kcat/Km. This is also called the specificityconstant and incorporates the rateconstants for all steps in the reaction(affinity and catalytic ability).
  25. 25. CO-ENZYMES Essential for Biological activity. Low molecular weight, Organic in nature Non protein in nature. .Combine loosely with Enzyme &separate later. Thermos table. Help in group transfer. Bind to apoenzymes. GTP, NADP, FMN, FAD, Biotin, Lipoic Acid, Pyridoxal Phosphate,etc. (Vitamins) Co-enzyme separate from apo-Enz after reaction. Can be separated by Dialysis.
  26. 26. Co-Enzymes can be divided into two groups.A.Oxidoreductases.NADH.NAD PH,FAD.B. Transfer Groups.Thiamine-Hydroxyl group.Pyridoxal phosphate-Amino groupTetrahydrofolate-one CarbonBiotin-Carbon dioxide .
  27. 27. Control Points of Gene Regulation Transcription RNA Processing DNA DNA RNA Transport 5’ mRNA RNA Degradation process 3’ ribosome maturemRNA mRNA Translation cap 5’ 3’ proteins tail Activity proteins ProteolysisProkaryotics Post-translational Eukaryotics control Juang RH (2004) BCbasics
  28. 28. Enzyme structure Enzymes are proteins They have a globular shape A complex 3-D structure Human pancreatic amylase© 2007 Paul Billiet ODWS
  29. 29. STRUCTURE1.MONOMERIC: Single Peptide.2.OLIGOMERIC: Many peptide Chains.3.Multienzyme Complex: Fatty Acid Synthase LDH Complex. Prostaglandin Synthase
  30. 30. ENZYMES UNITSKINGARMSTRONG.SOMOGY.REITMAN FRANKEL.SPECTROPHOTOMETRIC.KATAL.INTERNATIONAL UNIT.
  31. 31. ENZYMEZS ESTIMATED FROM:WHOLE BLOOD, SERUM, PLASMA.RED BLOOD CELLS.C.S.F.URINE.SWEAT.SALIVA.SEMEN.AMNIOTIC FLUID.Tears.
  32. 32. TISSUESBRAIN,HEART,LIVER,KIDNEY,MUSCLE BRAIN MUSCLE → ← ← HEART → ← KIDNEY ← LIVER STOMACH INTESTINE
  33. 33. PLASMA ENZYMES FUNCTIONAL PLAMSMA ENZYMES. eg. LIPOPROTEIN LIPASE, BLOOD CLOT DISSOLVING ENZYMES etc. NONFUNCTIONAL PLASMA ENZYMES. eg: SGOT, SGPT,AMYLASE,CPK,LDH,LIPASE,ACID -PHOSPHATASE,ALKALINE PHOS., CERULOPLASMIN etc.
  34. 34. NATURE OF ENZYMES Soluble,Colloidal, Organic Catalysts Formed by Living Cells ,Specific in action, Protein In Nature ,Inactive at Zero degree centigrade ,Destroyed by moist heat at 100 degree centigrade (Heat Labile), Huge in size, small Active Site, Used for Treatment.
  35. 35. DIFFERENCE BIO-CATALYST : Enzymes, protein in nature except ribozymes, More specific, more efficient and slight change in structure alter its action. CATALYST: Inorganic, less sp., less efficient and no change in structure.
  36. 36. Compartments of cell DNA, RNA, protein overviewDNARNAMutationsAmino acids,protein structure
  37. 37. COMPARTMENTATION MITOCHONDRIA: Enzymes of: E.T.C, TCA Cycle, Beta Oxidation, Urea Cycle, Pyruvate to Acetyle Co-A. CYTOSOL: Glycolysis, HMP Shunt, Fatty Acid Synthesis, Glucogenesis and Glycogenolysis. NUCLEUS: DNA Synthesis, RNA Synthesis and Histones etc. LYSOSOMES : Next Slide
  38. 38. FUNCTIONS OF ENZYMES 1. Catalyse thousands of reactions. 2. Digestive Enzymes help in igestion. 3. Lysosomal Enzymes destroy in cell. 4. Lysozymes are bacteriocidal, local immunity (TEARS)4. Detergents5. Textile.6. Leather Industry.
  39. 39. What is a Ribozyme?1) Enzyme2) Ribonucleic AcidNOT PROTEIN Sid Altman Tom Cech 1989 Nobel Prize In Chemistry
  40. 40. RIBOZYMESSmall ribonucleic particles.Contain rRNA.Highly substrate specific.Used in Intron splicing,pre RNA to RNA Peptidyl Transferase.Many ribozymes have hair-pin or hammer head shaped active centre &require Divalent Mg++Catalyse reaction on phosphpdiester bonds of other
  41. 41. Ribozymes Have following Drawbacks.Not as efficient as protein catalysts( In RNA there are 4 nucleotides, in amino acid are 20 in number.Act once only in chemical event,protein enzymes are reused several times.Rate of catalytic activity is slower.Synthatic Ribozymes are having better catalytic activity(Cleave infectious Virus)
  42. 42. ABZYMESArtificially synthasized catalytic antibodies against Enz. Sub. Complex in transition state of reaction. CATMAB (Catalytic Monoclonal Antibody).Sometimes natural abzymes are found in blood,eg.antivasoactive intestinal peptide autoantibodies.Useful in diseases viz.abzyme against gp120 envelop protein of HIV may prevent virus entry in to the host
  43. 43. ANTIENZYMEExtracts of Intestinal Parasites like Ascaris,contain substances called antizymes,which inhibit the action of digestive enzymes pepsin and Trypsin.This is probably the reason why the worms are not digested in the
  44. 44. PheRibozyme vs. tRNA folding
  45. 45. The Future of Ribozymes In Vitro Molecular Evolution of RNA + High Throughput Screening Ribozyme-Based Therapies
  46. 46. In Clinical Trial...HIV Gene Therapy... Bone Marrow Sample Treat Stem Cells with Retroviral Vector Encodes Gene for anti-HIV Ribozyme Re-Implant Treated Cells
  47. 47. ACTIVE SITE OF RIBONUCLEASESIt lies in a hydrophobic cleft.7 th Lysine 41 st Lysine on one side and 12 th Histidine and 119Histidine on the opposite side.(URIDYLIC ACID)Peptidyl transferase (chain Elongation)Removal of Introns.
  48. 48. The Substrate The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site© 2007 Paul Billiet ODWS
  49. 49. PRODUCT Substrate in the presence of Enzyme is converted in to product. The reaction can be Reversible or Ir- reversible. The increase in product concentration can cause inhibition and stop the reaction in the forwaed direction.
  50. 50. ABBREVIATIONS ENZYME [E] SUBSTRATE [S] PRODUCT [P] Enz. Sub. Complex [ES] INHIBITOR [I] Enz.+Inh. Complex [EI ] Enz.+Sub.+Inh. [ESI]
  51. 51. Enzymes are Biological Catalysts
  52. 52. Enzymes are proteins that:Increase the rate of reaction bylowering the energy of activation.Catalyze nearly all the chemicalreactions taking place in the cellsof the body.Have unique three-dimensionalshapes that fit the shapes of
  53. 53. Enzyme Deficiency A variety of metabolic diseases are nowknown to be caused by deficiencies ormalfunctions of enzymes. Albinism, for example, is often causedby the absence of tyrosinase, an enzymeessential for the production of cellularpigments. The hereditary lack of phenylalaninehydroxylase results in the diseasephenylketonuria (PKU) which, if untreated,leads to severe mental retardation in children.
  54. 54. ACTIVE SITEThe active site :Is a region within an enzyme that fits the shape of molecules called substrates .Contains amino acid R groups that align and bind the substrate.Releases products when the reaction is complete.
  55. 55. ACTIVE SITE OF ENZYMEChymotrypsin His(57)Asp(102)Ser(195)Trypsin Histidine,SerinePhosphoglucomutase SerineCarboxypeptidase Histidine,Arginine,tyrosineAldolase Lysine
  56. 56. Active Site Avoids the Influence of Water + -Preventing the influence of water sustains
  57. 57. Active Site Is a Deep Buried Pocket Why energy required to reach transition state is lower in the active site? It is a magic pocket + (1) Stabilizes transition (2) Expels waterCoE (1) (2) (3) Reactive groups(4) - (4) Coenzyme helps (3) Juang RH (2004) BCbasics
  58. 58. ACTIVE SITE Generally the active site is situated on the cleft of the Enzyme. Binding of substrate to active site dependends upon the presence of sp. Groups or atoms at active site. During binding these groups,realign themselves so as to fit the substrate. The substrate bind to active site by non co-valent bonds.(Hddrophobic in nature) Amino acid that make or break bonds called catalytic group.
  59. 59. ACTIVE SITE
  60. 60. EnzymesEnzymes provide a site where reactants can be brought together to react.Such a site reduces the energy needed for a reaction to occur.
  61. 61. All enzymes have an active site, where substrates are attracted to. Enzymes are used over and over again.
  62. 62. The Enzyme Substrate complex When enzymes function the active site interacts with the substrate. The active site shape matches the substrates shape. Once the substrate and active site meet a change in shape of the active site causes a stress that changes the substrate and produces an end product.
  63. 63. Which one will fit ?
  64. 64. ENZYME SPECIFICITYEnzymes may recognize and catalyze: A single substrate. A group of similar substrates. A particular type of bond.
  65. 65. MECHANISM OF ACTION INDUCEFIT MODEL. (KOSHLAND’S) LOCK AND KEY MODEL. (FISHER’S TEMPLATE THEORY)
  66. 66. The Induced Fit Hypothesis Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation T he active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction© 2007 Paul Billiet ODWS
  67. 67. Induced-fit ModelIn the induced-fit model of enzyme action: The active site is flexible, not rigid. The shapes of the enzyme, active site, and substrate adjust to maximum the fit, which improves catalysis. There is a greater range of substrate specificity.
  68. 68. The Lock and Key Hypothesis Fit between the substrate and the active site of the enzyme is exact Like a key fits into a lock very precisely The key is analogous to the enzyme and the substrate analogous to the lock. Temporary structure called the enzyme- substrate complex formed Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate© 2007 Paul Billiet ODWS
  69. 69. Lock-and-Key ModelIn the lock-and-key model of enzyme action: The active site has a rigid shape. Only substrates with the matching shape can fit. The substrate is a key that fits the lock of the active site. Rigid structure could not explain flexibility shown by enzymes
  70. 70. Acid-Base Catalysis Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.167 Specific Induced to transition state Acid-base Acid Catalysis + catalysis + N N O H H O = C H O O = + C H = = N C Both H H C H N C C H N C H H N C H H O H O H - O H H H H O H H H H O - O O - O Base C C catalysis Slow Fast Fast Very Fast
  71. 71. Basic Mechanism of Catalysis 3 basic types 1) Bond Strain Conformational change 2) Acid-base transfer Chemical reaction 3) Orientation Space arrangementConcert Carboxypeptidase A non-polar Metal protease Carboxypeptidase B RK Exopeptidase Carboxypeptidase Y non-specificSequential Chymotrypsin YFW Ser-protease Trypsin RK Endopeptidase Elastase GA Juang RH (2004) BCbasics
  72. 72. Concerted Mechanism of Catalysis Carboxypeptidase AActive (248) (270) site Tyr Glu 3 4 ACTIVEpocket O - Site for COO - H SITE specificity H + H O- C N R 5 N C C 2Substrate O- Juang RH (2004) BCbasics peptide chain 1 + COO - + Arg (145) His (196) Zn Glu C-terminus (72) Check for His (69) C-terminal
  73. 73. MICHAELI SCONSTAN (Km) T
  74. 74. Salient Features of Km Km is sub. Conc.at ½ the max. velocity It denotes that 50% of Enzyme mol.are bound with sub.at particular sub. Conc. Km is independent of Enzyme conc.If Enz. Conc. Is doubled, the Vmax will be double but km will remain same. Km is signature of Enzyme. Affinity of Enz. Towards its substrate is inversely related to the dissociation constant(smaller the dissociation greater the affinity. Km denotes affinity of enzme for substrate.lesser the Km more the affinity.
  75. 75. MICHAELIS CONSTANT (Km)It is defined as the conc. Of the substrate at which the reaction velocity is half of the maximum velocity.Km is independent of enzyme conc.If an enzyme has a small value of Km, it achieves maximal catalytic efficency at low substrate conc.SIGNIFICANCEGlucokinase has high Km is low
  76. 76. Hexokinase has low Km, High affinity for Glucose ie glucose willbe provided to the vital organs even at low glucose levels.Lab. Significance: The sub. Conc. Kept at saturation point at least 10 times the Km so that reaction proceeds to completion.Clinical Significance: The Km value for the given enzyme may differ from person to person and explains various responses to drugs/chemicals.
  77. 77. Names of EnzymesThe name of an enzyme: Usually ends in –ase. Identifies the reacting substance. For example, sucrase catalyzes the reaction of sucrose. Describes the function of the enzyme. For example, oxidases catalyze oxidation. Could be a common name, particularly for the digestion enzymes such as pepsin and trypsin .
  78. 78. The top-level classification is: EC 1 Oxidoreductases : catalyze oxidation/ reduction reactions . EC 2 Transferases : transfer a functional group (e.g. a methyl or phosphate group). EC 3 Hydrolases : catalyze the hydrolysis of various bonds . EC 4 Lyases : cleave various bonds by means other than hydrolysis and oxidation. EC 5 Isomerases : catalyze isomerization changes within a single molecule. EC 6 Ligases : join two molecules with covalent bonds.
  79. 79. CLASSIFICATION I.U.C.B. 1.OXIDO-REDUCTASE .transfer of hydrogen or addition of oxygen.Eg.LDH 2.TRANSFERASE. Eg.Aminotransferase. Hexokinase. 3.HYDROLASE .Cleave bond adding water Eg. Acetyl choline esterase. 4.LYASE .Cleave without adding water (Aldolase) 5.ISOMERASE. 6.LIGASE. Acetyl co-A carboxylase,Glu.Synthatase,PRPP Synthatase.
  80. 80. Classification of Enzymes:Oxidoreductases and Transferases
  81. 81. Classification: Hydrolases and Lyases
  82. 82. FACTORS AFFECTING ENZYME1.SUBSTRATE CONCENTRATION.2.ENZYME CONCENTRATION.3.TEMPERATURE.4.pH.5.EFFECT OF PRODUCT CONC.6.PRESENCE OF
  83. 83. FACTORS ………………..9.EFFECT OF CLOSE CONTCT.10.OXIDATION OF ADD.GROUPS.11.EFFECT OF LIGHT.12.EFFECTS OF RADIATIONS.13.PRESENCE OF REPRESSOR
  84. 84. Substrate concentration: Non-enzymic reactions Reaction velocity Substrate concentration The increase in velocity is proportional to the substrate concentration
  85. 85. Substrate Concentration The rate of reaction increases as substrate concentration increases (at constant enzyme concentration). Maximum activity occurs when the enzyme is
  86. 86. Substrate concentration: Non-enzymic reactions Reaction velocity Substrate concentration The increase in velocity is proportional to the substrate concentration
  87. 87. Substrate concentration: Enzymic reactionswhen[ s] conc. Is increased velocity increases in the initial phase (Vmax.),but flatten afterward. Vmax Reaction velocity Substrate concentration Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. If you alter the concentration of the enzyme© 2007 Paul BillietV max will change too. then ODWS
  88. 88. Enzyme Concentration The rate of reaction increases as enzyme concentration increases (at constant substrate concentration). At higher enzyme concentrations, more substrate binds with enzyme.
  89. 89. EFFECT OF CONC.PRODUCT At Equilibrium as per law of mass action,the reaction rate is slowed down,it can slow,stopped or reversed. A —E1—B —E2—≠— C —E3—D . Increase in conc. Of D will cause feed back Inhibition.
  90. 90. The effect of temperature For most enzymes the optimum temperature is about 30°C Many are a lot lower, cold water fish will die at 30°C because their enzymes denature A few bacteria have enzymes that can withstand very high temperatures up to 100°C Most enzymes however are fully denatured at 70°C© 2007 Paul Billiet ODWS
  91. 91. Affects of temperature on an enzyme If temp to high or to low the enzyme will not fit. No reaction will occur.
  92. 92. Temperature and Enzyme ActionEnzymes : Are most active at an optimum temperature (usually 37°C in humans). Show little activity at low temperatures. Lose activity at high temperatures as denaturation occurs.
  93. 93. The effect of temperature Q10 Denaturation Enzyme activity 0 10 20 30 40 50 Temperature / °C
  94. 94. The effect of temperature Q10 (the temperature coefficient ) = the increase in reaction rate with a 10°C rise in temperature. For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature) Enzyme-controlled reactions follow this rule as they are chemical reactions BUT at high temperatures proteins denature The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.
  95. 95. Optimum pH Values Most enzymes of the body have an optimum pH of about 7.4. In certain organs, enzymes operate at lower and higher optimum pH values.
  96. 96. The effect of pH Extreme pH levels will produce denaturation The structure of the enzyme is changed The active site is distorted and the substrate molecules will no longer fit in it At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur This change in ionisation will affect the binding of the substrate with the active© 2007 Paul Billiet ODWS
  97. 97. How pH affects an enzyme Ifthe pH is to high or low the enzyme will not work, because its shape will change.
  98. 98. The effect of pH Optimum pH values Enzyme activity Trypsin Pepsin 1 3 5 7 9 11© 2007 Paul Billiet ODWS pH
  99. 99. pH and Enzyme ActionEnzymes : Are most active at optimum pH. Contain R groups of amino acids with proper charges at optimum pH. Lose activity in low or high pH as tertiary structure is disrupted.
  100. 100. Optimum pH Values Most enzymes of the body have an optimum pH of about 7.4. In certain organs, enzymes operate at lower and higher optimum pH values.
  101. 101. ENZYME ACTIVATION BY INORGANIC IONSIn the presence some inorganic ions some enzymes show higher activity eg.Chloride ion activate salivary amylase,Ca. activates lipases.Proenzymes in to enzymes.Coagulatio factors are seen in blood as zymogen.Compliment cascade,these activities needed occasionly.
  102. 102. Enzyme InhibitionCompetitive Inhibtion.Non-Competitive Inhibition.Un-competitive Inhibition.Suicide Inhibition.Allosteric InhibitionKey EnzymesFeedback Inhibition.Inducors.Glucokinase is induced by Insulin.Repression (Heme is reprossor of ALA Synthase.
  103. 103. Enzyme Inhibition (Mechanism) I Competitive I Non-competitive I Uncompetitive Substrate ECartoon Guide S S E I S X E S I I I Compete for S I Inhibitor active site Different site E + S ← ES → E + P → E + S ← ES → E + P → E + S ← ES → E + P →Equation and Description + + + + I I I I ↓↑ ↓↑ ↓ ↑ ↓↑ EI EI + S →EIS E IS [I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex and competes with [S]; complex; Increasing [S] can only, increasing [S] favors increasing [S] overcomes not overcome [I] inhibition. the inhibition by [I]. Inhibition by [I]. Juang RH (2004) BCbasics
  104. 104. Competitive InhibitionProduct Substrate Competitive Inhibitor Succinate Glutarate Malonate OxalateC-OO- C-OO- C-OO- C-OO- C-OO-C-H H-C-H H-C-H H-C-H C-OO-C-H H-C-H H-C-H C-OO-C-OO- C-OO- H-C-H C-OO- Succinate Dehydrogenase
  105. 105. Sulfa Drug Is Competitive Inhibitor Domagk (1939) Para-aminobenzoic acid (PABA) Bacteria needs PABA for H2N- -COOH the biosynthesis of folic acid Folic Tetrahydro-Precursor acid folic acid Sulfa drugs has similar H2N- -SONH2 structure with PABA, and inhibit bacteria growth. Sulfanilamide Sulfa drug (anti-inflammation)
  106. 106. Enzyme Inhibitors Are Extensively Used● Sulfa drug (anti-inflammation) Pseudo substrate competitive inhibitor● Protease inhibitor Alzheimers disease Plaques in brain contains protein inhibitor● HIV protease is critical to life cycle of HIV HIV protease (homodimer): subunit 1 subunit 2↑inhibitor is used to treat AIDS Asp Asp Symmetry → Human aspartyl protease: domain 1 domain 2 Not symmetry (monodimer) Asp Asp
  107. 107. Signal Transduction Network (Ras vs. P53)SignalCell function are controlled by protein interactions Receptor Cell membrane Cytosol Regulator protein Ras Signal protein Effector Transcription enzyme Ribosome Inhibitor Apoptosis Nucleus P53 E2F mRNA Transcription factor Target gene Transcription mRNA Cell division ON Juang RH (2007) BCbasics
  108. 108. GP kinase Phosphatase GP b GP kinase P GP a Glycogen synthase Glucagon Glycogen Glycogen synthase P A PKA Protein phosphatase-1 Protein phosphatase-1 P Protein phosphatase inhibitor-1active Protein phosphatase inhibitor-1 Pinactive
  109. 109. Classification of Proteases  Family Example   Mechanism Specificity Inhibitor 2+Metal Carboxy- Zn H196 Non- EDTA E72 H69Protease peptidase A polar EGTA Aromatic DFP S195-O-Serine Chymotrypsin H57 TLCKProtease Trypsin D102 Basic TPCK C25-S-Cysteine Non- PCMB Papain H195 specific LeupeptinProteaseAspartyl Pepsin D215 Non- H2O specific PepstatinProtease Renin D32 Juang RH (2004) BCbasics
  110. 110. voSigmoidal Curve Effect Noncooperative (Hyperbolic) Positive effector ATP (ATP) Sigmoidal curve CTP brings sigmoidal curve Cooperative back to hyperbolic (Sigmoidal) Negative effector (CTP) keeps vo Exaggeration of sigmoidal curve yields a drastic Consequently, zigzag line that Allosteric enzyme shows the On/Off can sense the point clearly concentration of the environment and adjust its activity Off On [Substrate] Juang RH (2004) BCbasics
  111. 111. INDUCTIONInduction is effected through the process of derepression.The inducer will relieve the repression on the operator site.In the absence of glucose,the enzymes of Lactose metabolism will increase thousand times.Insulin is Inducer of Hexokinase Enzyme.Barbiturates induce ALA Synthase.
  112. 112. REPRESSIONInhibition and repression reduce the Enzyme Velocity.In case of Inhibition the Inhibitor act directly on the Enzyme.Repressor acts at the gene level,effect is noticed after a lag period of Hours or Days.
  113. 113. CO VALENT MODIFICATIONActivity of Enzyme can be increased or decreasd by co-valent modifications Eg.Either addition of group or Removal of groupZymogen activation by partial proteolysis is an Eg. Of co-valent modification
  114. 114. ADP RIBOSYLATION It is a type of co-valent modification. ADP-Ribose from NAD is added to enzyme/Protein. ADP Ribosylation of Alfa Sub unit of G Protein leads to Inhibition of GTPase activity;hence G protein remains active. Cholera toxin & Pertussive toxin act through ADP-Ribosylation. ADP Ribosylation of Glyeraldehyde 3P- Dehydrosense,result in inhibition of glycolysis.
  115. 115. Regulation of Enzyme ActivityInhibitor Proteolysis or proteolysis o I I x x o S I I S inhibitorFeedback regulation Phosophorylation o R x x P o S R S P (+) regulator effector phosphorylationSignal transduction Juang RH (2004) BCbasics A or A x o + Regulatory cAMP or S subunit calmodulin (-)
  116. 116. REGULATION OF ENZYMES‘’The action of enzymes can be activated or inhibited so that the rate of enzyme productin responds to the physiologcal need of the cell done to achieve cellular economy’’
  117. 117. 1. Allosteric Regulation.2. Activation of Latent Enzyme.3. Comprtmentation of Enzymes of different Pathways.4. Control of Enzyme Synthesis.5. Enzyme Degradation.
  118. 118. CHANGE IN CATALYTIC EFFICIENCY OF ENZYME Catalytic effeciency is regulated is modulated by A. Allosteric Regulation. B. Covalent modification . A. ALLOSTERIC REGULATION: Here the site is different from substrate binding site, this site is called ALLOSTERIC SITE. Low molecular wt. substances bind at site other than catalytic site,these are called ALLOSTERIC MODULATORS.Location is called allosteric site.
  119. 119. Examples of Second messengers are cAMP,cGMP and calcium etc.These can change the enzyme conformation that may alter either Km or Vmax.Based on this effect they are classified in to two
  120. 120. cAMP Controls Activity of Protein Kinase A Regulatory A A Active kinase subunits A cAMP A A C R C R Alberts et al (2002) Molecular Biology of the Cell (4e) p. 857, 858 A A R C Catalytic R C A subunitsNucleus CREB Activation P C Gene CREB expression DNA ON
  121. 121. EXAMPLE OF 2 nd MESSENGERGLYCOGEN GLYCOGEN BREAKDOW SYNTHESIS. N.
  122. 122. A.Activator A.Inhibitor . Allosteric  AllostericInhibitor . Activator .  Glucose-6-P,ATP Hexokinase: ADP  Glucose-6-P,ATP Isocit.Dehydr. ADP  ATP, NADH. Glu.Dehy. ADP Pyruvate Carboxylase  ADP Acetyl CoA
  123. 123.  HOMOTROPIC EFFECT : If the effector Substace is substrate itself it is called homotropic effect. HETEROTROPIC EFFECT : Effector molecule is a substance other than substrate. SECOND MESSENGER:Binding of many hormones to their surface receptor induce a change in enzyme catalysed reaction by inducing the release of allosteric effector.These effector substances are called as 2 nd messenger Hormone is first messenger. Cont……
  124. 124. CONFERMATIONAL CHANGES IN ALLOSTERIC ENZYMES . Most of Enzymes are oligomeric, binding of effector moecule at the allosteric site brings a chage in the active site of enzyme leading to inhibition or activation. Allosteric Enzyme exhist in two states. A. Tense (T) B. Relaxed (R ) Both are in equlibrium.
  125. 125. Allosteric Enzyme ATCase Active relaxed form Carbamoyl Aspartate Carbamoyl aspartate phosphate COO- COO- - - - - - - O CH2 O CH2 + = =H2N-C-O-PO32- HN-C-COO- H2N-C- N-C-COO- ATCase - - HH HHQuaternary structure ATP Feedback CCC Catalytic subunits inhibition CTP CTP CTP R R R CTP Regulatory subunits CTP CTP CTP R R R CCC Catalytic subunits Nucleic acid Inactive tense form Juang RH (2004) BCbasics metabolism
  126. 126. The switch: Allosteric inhibition Allosteric means “other site” Active site E Allosteric site© 2008 Paul Billiet ODWS
  127. 127. The allosteric site the enzyme “on-off” switch Active site E AllostericSubstrate site empty Conformational Efits into change Inhibitorthe active molecule Substratesite is present cannot fit The inhibitor into the molecule is active site Inhibitor fits absent into allosteric site© 2008 Paul Billiet ODWS
  128. 128. The Reception and Transduction of Signals Gilman, Rodbell (1994) G-protein-linked Receptor Glucagon Adenylate cyclase + Signal     -GDP     GTP GTP GDP GDP +GTP G protein A Glycogen breakdownThe third group: Insulin Enzyme-linked ReceptozIon-channel-linked Receptor  + Signal Juang RH (2007) BCbasics Glycogen Activation P kinase P  Synthase P P Protein active Phosphatase P P Glycogen SH2 Synthase Glycogen domain
  129. 129. voSigmoidal Curve Effect Noncooperative (Hyperbolic) Positive effector ATP (ATP) Sigmoidal curve CTP brings sigmoidal curve Cooperative back to hyperbolic (Sigmoidal) Negative effector (CTP) keeps vo Exaggeration of sigmoidal curve yields a drastic Consequently, zigzag line that Allosteric enzyme shows the On/Off can sense the point clearly concentration of the environment and adjust its activity Off On [Substrate] Juang RH (2004) BCbasics
  130. 130. FEED BACK INHIBITION Enzyme is inhibited by end product of reaction. A-B-C-D-E-F……….P. P product will inhibit the enzyme which converts A in to B.
  131. 131. COVALENT MODIFICATIONS Two well known processes A. PHOSPHORILATION. B. PARTIAL PROTEOLYSIS. A. Phosphorilation- dephosphorilation:many enzymes are regulated by ATP dependent phosphorilation.Eg. Of Serine,Threonine,and tyrosine,catalysed by protein kinases.
  132. 132. PARTIAL PROTEOLYSIS Some enzymes are secreted as inactive precursors called Proenzymes or Zymogens. This convertion takes place as a selective proteolysis. It is ir-reversible process Pepsinogen to pepsin Trypsinogen to trypsin.
  133. 133. Hexokinase have low KM High affinity for Glucose ie glucose will provide to the vital organs even at low glucose levels.Lab. Significance: The sub. Conc. Kept at saturation point at least 10 times the Km so that reaction proceeds to completion.Clinical Significance: The Km value for the given enzyme may differ from person to
  134. 134. INHIBITORS
  135. 135. Inhibitors Inhibitors are chemicals that reduce the rate of enzymic reactions. The are usually specific and they work at low concentrations. They block the enzyme but they do not usually destroy it. Many drugs and poisons are inhibitors of enzymes in the nervous system. © 2007 Paul Billiet ODWS
  136. 136. The effect of enzyme inhibition  Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories A. Competitive Inhibition. B. Non Competitive Inhibition.© 2008 Paul Billiet ODWS
  137. 137. COMPETITIVE INHIBITION There is close structural resemblance of Inhibitor with the Substrate. Example: 1.Malonate ions Inhibit Succinate Dehydrgenae. 2.Xanthene Oxidase is inhibited by Allopurinol.
  138. 138. The effect of enzyme inhibition 2. Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site Examples  Cyanide combines with the Iron in the enzymes cytochrome oxidase  Heavy metals, Ag or Hg , combine with –SH groups. These can be removed by using a chelating agent© 2008 Paulsuch as EDTA Billiet ODWS
  139. 139. The effect of enzyme inhibition  Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase© 2008 Paul Billiet ODWS
  140. 140. Enzyme Inhibition (Mechanism) I Competitive I Non-competitive I Uncompetitive Substrate ECartoon Guide S S E I S X E S I I I Compete for S I Inhibitor active site Different site E + S ← ES → E + P → E + S ← ES → E + P → E + S ← ES → E + P →Equation and Description + + + + I I I I ↓↑ ↓↑ ↓ ↑ ↓↑ EI EI + S →EIS E IS [I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex and competes with [S]; complex; Increasing [S] can only, increasing [S] favors increasing [S] overcomes not overcome [I] inhibition. the inhibition by [I]. Inhibition by [I]. Juang RH (2004) BCbasics
  141. 141. Enzyme Inhibition (Plots) I Competitive I Non-competitive I Uncompetitive Vmax Vmax Vmax vo voDirect Plots Vmax’ Vmax’ I I I Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM Vmax unchanged Vmax decreased Both Vmax & Km decreased Km increased Km unchangedDouble Reciprocal 1/vo I 1/vo I 1/vo I Two parallel Intersect lines at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax at X axis 1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S] Juang RH (2004) BCbasics
  142. 142. Competitive InhibitionProduct Substrate Competitive Inhibitor Succinate Glutarate Malonate OxalateC-OO- C-OO- C-OO- C-OO- C-OO-C-H H-C-H H-C-H H-C-H C-OO-C-H H-C-H H-C-H C-OO-C-OO- C-OO- H-C-H C-OO- Succinate Dehydrogenase
  143. 143. Applications of inhibitors  Negative feedback : end point or end product inhibition  Poisons snake bite, plant alkaloids and nerve gases  Medicine antibiotics, sulphonamides, sedatives and stimulants© 2008 Paul Billiet ODWS
  144. 144. Enzyme pathways Cell processes (e.g. respiration or photosynthesis) consist of series of pathways controlled by enzymes eA eB eC eD eF A B C D E F Each step is controlled by a different enzyme (eA, eB, eC etc) This is possible because of enzyme specificity© 2008 Paul Billiet ODWS
  145. 145. End point inhibition  The first step (controlled by e A ) is often controlled by the end product (F )  Therefore negative feedback is possible A eA B eB C eC D eD E eF F Inhibition  The end products are controlling their own rate of production  There is no build up of intermediates (B, C, D and E)© 2008 Paul Billiet ODWS
  146. 146. ATP is the end point  This reaction lies near the beginning of the respiration pathway in cells  The end product of respiration is ATP  If there is a lot of ATP in the cell this enzyme is inhibited  Respiration slows down and less ATP is produced  As ATP is used up the inhibition stops and the reaction speeds up again© 2008 Paul Billiet ODWS
  147. 147. The switch: Allosteric inhibition Allosteric means “other site” Active site E Allosteric site© 2008 Paul Billiet ODWS
  148. 148. Switching off  These enzymes have two receptor sites  One site fits the Inhibitor substrate like Substrate molecule other enzymes cannot fit into  The other site fits the active site Inhibitor fits into an inhibitor allosteric site molecule© 2008 Paul Billiet ODWS
  149. 149. The allosteric site the enzyme “on-off” switch Active site E AllostericSubstrate site empty Conformational Efits into change Inhibitorthe active molecule Substratesite is present cannot fit The inhibitor into the molecule is active site Inhibitor fits absent into allosteric site© 2008 Paul Billiet ODWS
  150. 150. A change in shape  When the inhibitor is present it fits into its site and there is a conformational change in the enzyme molecule  The enzyme’s molecular shape changes  The active site of the substrate changes  The substrate cannot bind with the substrate© 2008 Paul Billiet ODWS
  151. 151. Negative feedback is achieved  The reaction slows down  This is not competitive inhibition but it is reversible  When the inhibitor concentration diminishes the enzyme’s conformation changes back to its active form© 2008 Paul Billiet ODWS
  152. 152. Phosphofructokinase  The respiration pathway accelerates and ATP (the final product) builds up in the cell  As the ATP increases, more and more ATP fits into the allosteric site of the phosphofructokinase molecules  The enzyme’s conformation changes again and stops accepting substrate molecules in© 2008 Paul Billiet ODWS
  153. 153. Competitive InhibitionProduct Substrate Competitive Inhibitor Succinate Glutarate Malonate OxalateC-OO- C-OO- C-OO- C-OO- C-OO-C-H H-C-H H-C-H H-C-H C-OO-C-H H-C-H H-C-H C-OO-C-OO- C-OO- H-C-H C-OO- Succinate Dehydrogenase Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49
  154. 154. Sulfa Drug Is Competitive Inhibitor Domagk (1939) Para-aminobenzoic acid (PABA) Bacteria needs PABA for H2N- -COOH the biosynthesis of folic acid Folic Tetrahydro-Precursor acid folic acid Sulfa drugs has similar H2N- -SONH2 structure with PABA, and inhibit bacteria growth. Sulfanilamide Sulfa drug (anti-inflammation)
  155. 155. Enzyme Inhibition (Plots) I Competitive I Non-competitive I Uncompetitive Vmax Vmax Vmax vo voDirect Plots Vmax’ Vmax’ I I I Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM Vmax unchanged Vmax decreased Both Vmax & Km decreased Km increased Km unchangedDouble Reciprocal 1/vo I 1/vo I 1/vo I Two parallel Intersect lines at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax at X axis 1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S] Juang RH (2004) BCbasics
  156. 156. The effect of enzyme inhibition Irreversible inhibitors : Combine with the functional groups of the amino acids in the active site, irreversibly.Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.© 2007 Paul Billiet ODWS
  157. 157. The effect of enzyme inhibition Reversible inhibitors : These can be washed out of the solution of enzyme by dialysis.There are two categories.© 2007 Paul Billiet ODWS
  158. 158. The effect of enzyminhibition1. Competitive : These compete with the substrate E+I EI molecules for the active site. Revers EnzymeThe inhibitor’s action is ible inhibitor proportional to reactio complex its concentration. nResembles the substrate’s structure closely.© 2007 Paul Billiet ODWS
  159. 159. CLINICAL APPLICATIONS OF COMPETITVE INHIBITORSDRUG ENZYME TRUE Clinical SUB. App.ALLOPURI XANTHINE HYPOXAN GOUTNOL OXIDASE THENESULFONA Dihydro PABA ANTIBIOTIMIDE pteroate C SynthaseETHANOL Al.Dehy. METHANO METHANO L L POISONIN G
  160. 160. NON-COMPETITIVE2. Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site.Examples Cyanide combines with the Iron in the enzymes cytochrome oxidase. Heavy metals, Ag or Hg, combine with – SH groups.These can be removed by using a chelating agent such as EDTA.© 2007 Paul Billiet ODWS
  161. 161. Applications of inhibitors Negative feedback : end point or end product inhibition Poisons snake bite, plant alkaloids and nerve gases. Medicine antibiotics, sulphonamides, sedatives and stimulants © 2007 Paul Billiet ODWS
  162. 162. ISOENZYMES ORISOZYMES
  163. 163. ISOENZYMES Isoenzymes or Isozymes are physically distinct form of same enzyme having same specificity, but are present in different tissues of same organism, in different cell compartment. Useful for diagnosing diseases of different organs. Homomultimer:All the units are same. Heteromultimer:Sub units are different.These are produced by different genes.
  164. 164. IDENTIFICATION OF ISOZYMES1.Agar gel or PAGE.They have different mobility.2.Heat stability.3.Inhibitors.Isozymes may be sensative to different inhibitors.eg.tartrate labile.4. Km value or substrate specificity. Eg.Glucokinase has high Km and Hexokinase has low Km for Glucose.
  165. 165. 5.Co-Factors.Eg Mitochondrialisocitrate dehydrogenase is NADdependent,Cytoplasmic isocitratedehydrogenase is NADP dependent.6. Localisation: LactateDehydrogenaseH4 heart,M4 Muscles.7.Specific antibodies identifysp.Isozyme.
  166. 166. Isoenzy Composit Compositi Present in Elevated inme ion onnameLDH1 ( H 4) HHHH Myocardiu myocardial m, RBC infarctionLDH2 (H 3 M 1 ) HHHM Myocardiu m, RBCLDH3 (H 2 M 2 ) HHMM Kidney, Skeletal muscleLDH4 (H 1 M 3 ) HMMM Kidney, Skeletal muscleLDH5 (M 4 ) MMMM Skeletal Skeletal muscle, muscle Liver and liver
  167. 167. Isoenzymes Isoenzymes catalyze the same reaction in different tissues in the body. Lactate dehydrogenase, which converts lactate to pyruvate, (LDH) consists of five isoenzymes.
  168. 168. Isoenz Composi Present Elevatedyme tion in inname CNSCK-1 BB Brain diseases Myocar AcuteCK-2 MB dium/ myocardi Heart al infarction SkeletalCK-3 MM muscle, Myocar
  169. 169. DIAGNOSTICSIGNIFICANC E
  170. 170. DISORDERS DIAGNOSED BY ENZYMES1) Cardiac Disorders. 4) Bone Disorders.2) Hepatic Disorders. 5) Pancreatic Disorders.3) Skeletal Muscle 6) Salivary gland Disorders. diseae (Mumps) 7) Malignancies
  171. 171. Plasma enzymes are of two types:1. A small group of enzymes secreted into the blood by certain cells e.g. the liver secretes zymogens (inactive form of enzymes) of blood coagulation.2. FUNCTIONAL: Lipoprotein lipase,Pseudocholine estrase,blood coagulation.3. NON FUNCTIONAL ENZYMES:
  172. 172. 2. A large group of enzymes are released from cells during normal cell turnover. These enzymes function intracellularly (inside cells) and have no function in the blood. In healthy individuals, the blood levels of these enzymes are constant, as the rate of release from damaged cells into blood is equal to the rate of removal of enzymes from blood.
  173. 173. Elevated enzyme activity in blood indicates tissue damage (due to increased release of intracellular enzymes).
  174. 174. A. Plasma Enzymes as diagnostic tools Diseases that cause tissue damage result in increased release of intracellular enzymes into the plasma. Determination of the level of these enzymes is used for diagnosis of heart, liver, skeletal muscle, etc. The level of these enzymes in plasma correlates with the extent of tissue damage.
  175. 175. CREATINE KINASE(CPK OR CK) Found in Heart, Skeltol Muscles,Brain small amounts are also found in lungs, thyroid and Adrenal glands. Not found in RBC so haemolysis no effect. NORMAL SERUM LEVELS: 10-50 IU/L at 30 degree Centigrade.
  176. 176. CREATINE KINSE…………………… RAISED LEVELS ARE FOUND IN : 1.Myocardial Infarction. 2. Crushing Muscular Injury. 3. Damage to cardiac muscle (Any region) 4. Brain Injury. 5. Hypothyroidism. 6. Hypokalemia Highest level in 3-6,peak 24-30 hours normaml in 3days.
  177. 177. ISOENZYMES OF (CPK) 1.BB (CPK1) Tissue. is of origin is Brain,Maximum Electrophoretic mobility, presence in blood is 0%. MB (CPK2) Found in heart muscles, Intermediate electrophoretic mobility, presence in blood is 0-3%. MM (CPK 3) Found in skeltol muscles,Least electrophoretic mobility, in blood its conc. Is 97-100 %.
  178. 178. Myocardial muscle is the only tissue that contains high level of CK2 (MB) isoenzyme.Appearance of CK2(MB) in plasma is specific for heart infarction.Following an acute myocardial infarction,CK2appears in plasma 4-8 hours following onset of chest pain (peak is
  179. 179. ASPARTATE AMINO TRANSFERASE AST (SGOT)SERUM GLUTAMATE OXALOACETATE TRANSFERASE NORMAL LEVELS: 0-41 IU/L Rises in 12 hours ,Peak levels 24 hours Returns to normal 3-5 days.
  180. 180. ALANINE TRANSAMINASE (ALT) ALT Highest conc. In Liver and next is skeltol muscles. Raised levels are found in liver diseases and muscle disorders. Marked elevation are found in acute hepatitis and other liver diseases.
  181. 181. The presence of increased levels of some enzymes in plasma is diagnostic to damage of a particular tissue; e.g. The enzyme alanine aminotransferase (ALT) is abundant in the liver and the appearance of elevated levels of ALT in plasma indicates damage to the liver.
  182. 182. ALKALINE PHOSPHATASE (ALP) Works at optimum pH 9. Highest conc. Are found in Liver, Bone,Intestine and Placenta. Diagnosis of Bone and Liver Pathology Metastatic or Primary Malignant may increase the enzyme activity. It has many Iso-enzymes. Cont…..
  183. 183. ISOENZYMES OF (ALP) 1. Alfa-1 ALP : Biliary canaliculi raised activity shows obstructive jaundice. 2. Alfa-2 ALP Its levels rises in Hepatitis. 3.Pre Beta ALP: Bone cells, Bone diseases raised levels are found. 4. Gama ALP :Found in Intestinal cells. Levels rise in Ulcerative colitis. 5. Distinct Type: levels rise in Lymphomas,Decrease in Chronic myl.Leuk. 6. Regan Isoenzyme:Cancer of lung,liver, Gut.
  184. 184. GAMA GLTAMYL TRANSPEPTIDASE (GGT) Itis a sensative indicator of liver diseases, especially of alcoholism. There are no other serum enzyme abnormalities.
  185. 185. ACID PHOSPHATE Exhists at pH 5-6. Diagnosis of Carcinoma of Prostate. Also found in RBC. Used as cancer Marker.
  186. 186. LACTATE DEHYDROGENASE (LDH) Enzyme of anaerobic glycolysis. Liver,Myocardium, RBC. It is a tetramer made up of four units. These units can be separated by electrolysis. There are two sub units (H&M)
  187. 187. Isoenzymes Isoenzymes catalyze the same reaction in different tissues in the body. Lactate dehydrogenase, which converts lactate to pyruvate, (LDH) consists of five isoenzymes.
  188. 188. LDH (ISOENZYMES) LDH 1 Tetramer of  Moves fastest at four units.30% in S. pH8.6,myocardium ,RBC LDH  Myocardium RBC . 2. 35% in Serum. LDH 3. 20%  Brain Kidney LDH 4 10%  Skeltol muscles ,Liver LDH 5 5%  ----- Do -----
  189. 189. AMYLASE / LIPASE Digestive enzymes,exocrine pancreas. Levels rise in Acute Pancreatitis. Patient present with severe abd. Pain. Lipase levels are raised in Intestinal infarction,Pertonitis or Perforation.
  190. 190. CHOLINESTRASE Secreted by hepatic cells. Always present in serum. Metabolism of drugs cocaine and succinyl- choline.
  191. 191. TRYPSINRaised levels of Trypsin in plasma occurs during acute stage of PANCREATITISAlong with Amylase and Lipase.It is a more reliable index of Pancreatic disease rather than Amylase/Lipase
  192. 192. Intracellular Distribution of Diagnostic EnzymesLiver Hea Pancre Saliva Bon Muscl Bilia Prosta rt as ry e e ry te Glands Trac tLD5 LD1 LPS AMS AL CK ALP ACPALT AST AMS P GGTAST CK
  193. 193. DISORDERS DIAGNOSED BY ENZYMES1) Cardiac Disorders. 4) Bone Disorders.2) Hepatic Disorders. 5) Pancreatic Disorders.3) Skeletal Muscle 6) Salivary gland Disorders. diseae (Mumps) 7) Malignancies
  194. 194. NAME OF THE Conditions in which ENZYME level of activity in serum is elevatedAspartate Amino Myocardial infarction,transferase (AST) Liver diseaseSerum glutamate- especially with liveroxaloacetate cell damagetransaminase(SGOT)Alanine Amino Liver diseasetransferase (ALT) especially with liverSerum glutamate- cell damage
  195. 195. Isoenzymes Isoenzymes catalyze the same reaction in different tissues in the body. Lactate dehydrogenase, which converts lactate to pyruvate, (LDH) consists of five isoenzymes.
  196. 196. Diagnostic Significance Enzymes The levels of diagnostic enzymes determine the amount of damage in tissues.
  197. 197. B. Isoenzymes and Heart Diseases Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction. However, these enzymes do not have the same physical properties (as they differ in amino acid sequence). Thus, they differ in electrophoretic mobility. The plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial infarction.
  198. 198. CARDIAC MARKERS CPK (MB) LDH (1) CARDIAC TROPONIN (I)&(T) BRAIN NATRIURETIC PEPTIDE (Marker of Ventricular function) AST ALT
  199. 199. Abnormal Liver enzymes and/or LFTs: work-up and diagnosis LIVER MARKERS
  200. 200. Liver Tests AST, ALT Alkaline Phosphatase GGT Bilirubin Albumin True “liver function tests” Protime/INR
  201. 201. AST, ALTAspartate aminotransferase, alanine aminotransferase Enzymes that are in the hepatocyte and function during gluconeogenesis Leak out of the hepatocytes in times of injury and can be measured in the serumNormally present in serum at levels ~30-40 U/L
  202. 202. Alkaline Phosphatase Exists in liver in membrane of hepatocyte where it lines the canaliculus Liver > bone > intestine Placenta Normally changes with age 400 350 300 250 200 150 100 50 5 15 25 35 45 55 65 75 85
  203. 203. Other cholestatic enzymes GGT: gamma-glutamyltransferase Found in hepatocytes and biliary epithelial cells 5’ nucleotidase Both these enzymes can be used to confirm alk phos elevation is coming from liver GGT is also sensitive to alcohol ingestion
  204. 204. Bilirubin Breakdown product of heme 70-80% of normal production is from breakdown of hemoglobin in senescent RBC Conjugation of bilirubin occurs in ER of hepatocyte, and conjugated bilirubin is then transported into bile (rate limiting step) Almost 100% of bilirubin in healthy people is indirect
  205. 205. Albumin Important plasma protein synthesized by the liver Half-life 20 days Levels <3 mg/dL should raise the suspicion of chronic liver disease ***not specific for liver disease Also reduced in heavy alcohol consumption, chronic inflammation, protein malnutrition
  206. 206. PROSTATE MAR PSA (prostate SP.ANTIGEN. ACP (Acid Phosphatase)
  207. 207. MUSCLE MARKER CK (MM) AST (Aspartate Amino Transferase) ALD (Aldolase)
  208. 208. BONE MARKER ALP (Alkaline Phosphatase)
  209. 209. 1.Cardiac Markers:e.g. Acute Myocardial Infarction (AMI).1) The myocardium becomes ischemic and undergoes necrosis.2) Cellular contents are released into the circulation. Blood levels of the following enzymes increase: AST LD1 CK
  210. 210. 2. Hepatic Disordersa) Hepatocellular Disorders: (1) Viral hepatitis: Hepatitis B & Hepatitis C. (2) Toxic hepatitis: caused by chemicals & Toxins (e.g aflatoxin, Asp. flavus) Increased levels of the following enzymes : ALT AST LD5
  211. 211. b) Biliary tract disorders: The plasma levels of the following enzymes increase: ALP GGT
  212. 212. 3. Skeletal Muscle Disorders Muscle dystrophy. Muscle trauma. Muscle hypoxia. Frequent I.M Injections. The plasma levels of the following enzymes increase: CK AST
  213. 213. 4. Bone Disorders:1) Paget’s Bone Disease: caused by increased osteoclastic activity.2) Rickets3) Osteomalacia: The plasma levels of the following enzyme increase: ALP
  214. 214. 5. Acute PancreatitisThe plasma levels of the following enzymes increase: Lipase AMS
  215. 215. 6. Salivary Gland Inflammation:In Mumps:The levels of α -Amylase (AMS) is significantly increased
  216. 216. 7. Malignanciesa) Plasma (Acid phosphatase) ACP levels increase in:• Prostatic carcinoma.• Bone metastatic carcinoma
  217. 217. b) Plasma levels of Alkaline phosphatase (ALP) increase in:• Pancreatic carcinoma.• Bile duct carcinoma.• Liver metastasis.
  218. 218. c) Plasma levels of Total Lactate dehydrogenase (LDH) increase in:• Leukemia• Lymphomas.• Liver metastasis.
  219. 219. ENZYMES TUMOUR MARKERS
  220. 220. B. Isoenzymes and Heart Diseases Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction. However, these enzymes do not have the same physical properties (as they differ in amino acid sequence). Thus, they differ in electrophoretic mobility. The plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial infarction.
  221. 221.  Many isoenzymes contain different subunits in various combinations. CK occurs in 3 isoenzymes, each is a dimer composed of 2 subunits (B & M): CK1 = BB, CK2 = MB and CK3 = MM, each CK isozyme shows a characteristic electrophoretic mobility.
  222. 222. Myocardial muscle is the only tissue that contains high level of CK2 (MB) isoenzyme.Appearance of CK2(MB) in plasma is specific for heart infarction.Following an acute myocardial infarction,CK2appears in plasma 4-8 hours following onset of chest pain (peak is
  223. 223. Alkaline Phosphatase1.Alfa1-ALP Liver2.Alfa2-ALP Liver (Heat Labile)3.Pre Beta-ALP (BONES)4.Gama ALP (Ulcerative Colitis)5.Regan ALP (Bronchogenic cancer)
  224. 224. ENZYMES IN OTHER BODY FLUIDSAdenosine deaminase in pleural fluid :Elevated in Tuberculosis not in Malignant effusion.LDH; In CSF,Pleural fluid & Ascitic Fluid.Elevated levels in Malignacy .
  225. 225. Enzymes as Therapeutic Agents Dissolving Streptokinase,Urokinase. Asparaginase used in some leukemias. Deoxyribonuclease is adminstered via respiratory route to clear viscid secretions in pt. of cystic fibrosis. Serratiopeptidase is used to minimise edema in acute inflamatory conditions. Hyaluronidase for hypovolumia Hemocoagulase used as hemostat.
  226. 226. ENZYMES USED INDIAGNOSTICS PROCEDURES Urease Urea. Uricase Uric Acid. Glucose Oxidase Glucose. Peroxidase Cholesterol. Hexokinase Glucose. Lipase Triglycerides. Alkaline phosphatase ELISA. Restriction endonuclease RFLP
  227. 227. Fungal Diastase &Pepsin 1. Clinical Enzymology Questions For a biological process to occur a free energy overcome. Enzymes work in this process to: a. Lower the free energy of activation of activation must beused as digestive enz. b. Raise the free energy of activation c. Enzymes have no effect on free energy o f activation d. The effect on free energy of activation is dependent on the enzyme in question e. None of the above 2. CK-M and CK-B are examples of what type of enzyme? a. Homogeneous enzymesRibozymes &Abzymes b. Isoenzymes c. Heterogeneous enzymes d. Co-factors e. None of the above 3. A 68-year-old male presents to the emergency room with acute mental confusion. Upon questioning his family members they recall that for the last several months he has been complaining of tingling and loss of feeling in his hands and feet, difficulty walking, and vomiting.Streptodornase; DNA Which of the following co -factors is he most likely suffering from a deficiency in? a. Folic Acid coenzymes b. Biotin c. Flavin coenzymes d. Thiamine pyrophosphate e. B12 coenzymesapplied locally. 4. Which of the following type of enzyme reaction does not normally require the use of a cofactor? a. Oxidation-reduction reaction b. Group Transfer reactions c. Isomerizations d. Hydrolytic reactionsAlpha-1-ant-trypsin;Emphysema

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