![Michaelis-Menten Equation
1. Reaction Model
2. Michaelis - Menten equation
Illustrates in mathematical term the relationship b/w
initial reaction velocity (Vi) & substrates
concentration [S].](https://image.slidesharecdn.com/enzymes-231006163650-0e29ad5c/85/ENZYMES-ppt-38-320.jpg)
![ Elevation of which of the following serum enzyme markers would be most useful in
diagnosing a myocardial infarction in a patient who comes to your office 3 days after
an episode of severe and prolonged substernal chest pain?
a. LDH isoenzymes b. CKMB c. Troponin I d. Myoglobin
e. Enzyme markers are no longer useful 3 days after an MI
When [S] is equal to Km, which of the following condition exists ?
a) Half of the enzyme molecules are bound to substrate.
b) The velocity of the reaction is equal to Vmax
c) Enzyme is completely saturated with substrate
d) The reaction has reached the equilibrium
"Lock& Key" model enzyme action proposed by Fischer implies that:
a) The active site is flexible& adjusts to substrate
b) The active site requires removal of PO4
c) The active site is complementary in shape to substrate.
d) All of them v. None of them
In competitive inhibition of enzyme action :
i. Apparent Km is decreased ii. Apparent Km is increased.
iii. Vmax is increased iv. Vmax is decreased
A person aged 48 years presented in CCU with history of retrosternal chest pain
radiating to left arm, breathlessness & sweating since 18 hours.
a) What is your possible diagnosis?
b) What enzymes would be raised in this patient?
c) Why are enzymes raised in certain functional diseases?
d) What other investigations will you suggest?
e) Along with treatment what will you advise this patient for future care?](https://image.slidesharecdn.com/enzymes-231006163650-0e29ad5c/85/ENZYMES-ppt-56-320.jpg)
ENZYMES.ppt
- 1. ENZYMES BY PROFESSOR DR. M. SHAFIQUE MBBS,M.Phil.,Ph.D
- 2. Enzymes Definition and Explanations Mechanism of action Classification Properties of enzymes Factors affecting enzyme activity Enzyme kinetics Regulatory enzymes Allosteric enzymes Enzymes of clinical interest
- 3. 3 What Are Enzymes? Most enzymes are Proteins (tertiary and quaternary structures) Act as Catalyst to accelerates a reaction Not permanently changed in the process
- 4. 4 Enzymes Are specific for what they will catalyze Are Reusable End in –ase -Sucrase -Lactase -Maltase
- 5. Definitions Enzyme: is a biological catalyst, synthesized by living cells, increases the velocity or rate of chemical reaction and is not consumed during that reaction. It is functional unit of cell metabolism. It is colloidal, thermo labile and mostly protein in nature. Holoenzyme = apoenzyme + coenzyme Coenzyme: is an organic cofactor which is thermo stable, non-proteinous, low molecular weight, required for the action of certain enzymes, it can be dialyzed out and separated from the enzyme. Many coenzymes are active forms of vitamin B-complexes Thiamine : TPP , TTP Riboflavin : FMN , FAD Niacin : NAD , NADP Pyridoxine: Pyr Phosphate Pentothenic acid : CoASH Vitamin B12 : Cobamide Folic Acid : FH4
- 7. 7 How do enzymes Work? Enzymes work by weakening bonds which lowers activation energy
- 8. 8 Enzymes Free Energy Progress of the reaction Reactants Products Free energy of activation Without Enzyme With Enzyme
- 9. 9 Enzyme-Substrate Complex The substance (reactant) an enzyme acts on is the substrate Enzyme Substrate Joins
- 10. 10 Active Site A restricted region of an enzyme molecule which binds to the substrate. Enzyme Substrate Active Site
- 11. 11 Induced Fit A change in the shape of an enzyme’s active site Induced by the substrate
- 12. 12 Induced Fit A change in the configuration of an enzyme’s active site (H+ and ionic bonds are involved). Induced by the substrate. Enzyme Active Site substrate induced fit
- 13. Mechanism of Action LOWER ACTIVATION ENERGY ENZYME SUBSTRATE COMPLEX Template or lock and key model Induced fit model CONVERSION OF SUBSTRATE TO PRODUCT (ES-------------EP) RLEASE OF PRODUCT FROM ENZYME The sequence is E + S----ES----EP----E + P
- 14. ENZYME ACTION
- 16. Lowering of the activation energy of Hydrogen peroxide Reaction condition Activation free energy (K.cal/mol) Relative rate No catlyst 18.0 1 Platinum surface 11.7 2.77×104 catalase 5.5 6.51×108
- 17. Classification of Enzymes I. Oxidoreductases II. Transferases III. Hydrolases IV. Lyases V. Isomerases VI. Ligases
- 18. I.Oxidoreductases :catalyze oxidation-reduction reactions Further divided into four subgroups Oxidases Dehydrogenases Hydroperoxidases Oxygenases 1. Oxidases Oxygen is added and H atoms are removed from the substrate forming H2O or H2O2 xanthine oxidase A.A oxidase
- 19. 2. Dehydrogenase They remove hydrogen from substrate, but are not able to use oxygen as hydrogen acceptor Special hydrogen acceptors such as NAD+, NADP+, FAD are used. Examples 1. Lactate dehydrogenase (LDH) 2. Succninate dehydrogenase 3. Glucose 6-Phosphate dehydrogenase 3. Hydroperoxidases Catalase and Peroxidase 2H2O2→2H2O+ O2
- 20. II. Transferases : Catalyze transfer of amino, phosphoryl, methyl, glycosyl groups 1.Transaminases – The catalyze the exchange of NH2 group between amino acid and keto acids. The keto acid become amino acid and amino acid become keto acid. e.g i. G.O.T (AST) ii. G.P.T (ALT) 2. Phaspho transferases: kinases 3. Transmethylases transfer of methyl group from methionine to form creatine & adrenaline etc.
- 21. 21 Transaminations Glutamate a-Ketoglutarate + + Pyruvate Alanine Glutamate a-Ketoglutarate + + Oxaloacetate Aspartate Glutamate-Pyruvate Aminotransferase(GPT) (Alanine Transferase ALT) Glutamate-Oxaloacetate Aminotransferase(GOT) (Aspartate Transferase AST) Blood levels of these aminotransferases, also called transaminases, are important indicators of liver disease
- 22. III. Hydrolases (catalyze hydrolysis ) 1. Protein hydrolyzing enzymes A. Exopeptidase: carboxypeptidase B. Endopeptidases: pepsin,trypsin,chymotrypsin,& elastase 2. Carbohydrases-: amylases, sucrase, lactase 3. Lipid hydrolyzing enzymes: lipases 4. Deaminases: adenase, guanase
- 23. LYASES Cause addition of NH3 ,H2O or CO2 to double bonds or removal of these from double bonds. FUMARIC ACID------------MALIC ACID ISOMERASES Cause isomerization of substrate, may be isomerases or mutases. GLUCOSE 6-P------------FRUCTOSE 6-P GLUCOSE 6-P------------GLUCOSE 1-P LIGASES Involve in joining together two substrates with help of ATP,GTP etc ACETYL CoA------------MALONYL CoA SUCCINIC ACID-------SUCCINYL CoA
- 25. ENZYME PROPERTIES SPECIFICITY Absolute Specificity Relative Specificity Reaction Specificity Substrate Specificity Stereo Specificity Bond Specificity PROTEIN NATURE ENZYME INHIBITION DIRECTION OF ENZYME REACTION PROENZYMES OR ZYMOGENS INDUCTION OF ENZYME SYNTHESIS REPRESSION OF ENZYMES ISOENZYMES
- 26. ISOENZYMES (ISOZYMES) These are multiple forms of an enzyme that differ in structure and properties but catalyze the same reaction. i. LACTATE DEHYDROGENASE:LDH ii. CREATININE KINASE:CK OR CPK iii. ALKALINE PHOSPHATASE:ALP LDH has five isoenzymes LDH1-LDH5.Each type has 4 polypeptide chains which are of two types M (muscle) and H (heart) M4 Chains: M4,M3H,M2H2,MH3 H4 Chains: H4,H3M,H2M2,HM3
- 27. ISOENZYMES LDH SUBUNITS LDH1-I1 HHHH LDH2-I2 HHHM LDH3-I3 HHMM LDH4-I4 HMMM LDH5-I5 MMMM CREATINE KINASE has 3 isozymes with 2 polypeptides B & M CK1-BB (Brain), CK2- MB (Myocardium),CK3-MM(Muscle) Ckmb is raised in myocardial infarction Alkaline phosphatase-ALP α1-ALP, α 2 ALP, pre-βALP & γ-ALP etc
- 28. 28 What Affects Enzyme Activity? Three factors: 1. Environmental Conditions 2. Cofactors and Coenzymes 3. Enzyme Inhibitors
- 29. 29 1. Environmental Conditions I. Extreme Temperature are the most dangerous - high temps may denature (unfold) the enzyme. II. pH (most like 6 - 8 pH near neutral) III. Ionic concentration (salt ions)
- 30. 30 2. Cofactors and Coenzymes Inorganic substances (zinc, iron) and vitamins (respectively) are sometimes need for proper enzymatic activity. Example: Iron must be present in the quaternary structure - hemoglobin in order for it to pick up oxygen.
- 31. 31 3. Examples of Enzyme Inhibitors a. Competitive inhibitors: are chemicals that resemble an enzyme’s normal substrate and compete with it for the active site. Enzyme Competitive inhibitor Substrate
- 32. 32 Inhibitors b. Noncompetitive inhibitors: Inhibitors that do not enter the active site, but bind to another part of the enzyme causing the enzyme to change its shape, which in turn alters the active site. Enzyme active site altered Noncompetitive Inhibitor Substrate
- 33. Enzyme and substrate concentrations
- 34. Effect of substrate concentration a) At low substrate concentration, the velocity of reaction is directly proportional to the substrate level (part A in graph). a) In the second phase (part B), the substrate conc. is not directly proportional to enzyme activity. c) In the third & final phase (part C), the reaction is independent of the substrate concentration.
- 35. Temperature and pH effect enzyme action
- 36. Temperature and pH effect enzyme action
- 38. Michaelis-Menten Equation 1. Reaction Model 2. Michaelis - Menten equation Illustrates in mathematical term the relationship b/w initial reaction velocity (Vi) & substrates concentration [S].
- 39. Michaelis-Menten Kinetics Shows saturation at high substrate concentrations Vmax – rate at saturation for a given enzyme concentration in moles per unit time Km – Michaelis constant – substrate concentration that gives ½ maximal velocity S K S V V m max
- 40. The equation of Lineweaver-Burk plot (Double reciprocal plot) The equation The plot
- 41. What is km ? The michaelis-Menten constant (Km) is the substrate conc. at which Vi is half maximal velocity (Vmax/2) attainable at particular concentration of enzymes. It indicates that half of the enzyme molecules(50%) are bound to substrates molecules when the substrates con. equals the Km value.
- 49. Allosteric enzyme (allo-other) Definition: Some of the enzymes possess addition sites, known as allosteric sites, besides the active sites. Allosteric effector or modulator .Positive allosteric effector .Negative allosteric effector
- 50. Drugs clinically used as inhibitors Naturally occurring substances Competitive inhibitors (anti - metabolites) Enzyme inhibits Chief action & use 1. p-Amino Benzoic Acid (PABA) Sulfonamides e.g Septran (sulfamethoxazole) Dihydro-pteroate synthetase Inhibit the formation of folic acid by the bacterial cell which is needed by them. The bacteria therefore die out. 2. Folic Acid Pteroylglutamate (PGA) Methotrexate (MTX) Metotrexate Dihydro-folate reductase (FH2 reductase) Inhibit the formation tetrahydro-folic acid, which is needed for DNA synthesis. Used as anti-cancer drug. 3. Hypoxanthine and xanthine Allopurinol (zyloric) Xanthine Oxidase (purine catabolism) Inhibit formation of uric acid; used in treatment of gout.
- 51. Uses of Enzymes 1. For diagnosis & prognosis of diseases by its estimation in serum. i.e Cardiac enzymes: CK, AST, LDH, Troponin T & Troponin I (cTnI) Liver enzymes: Transaminases, Alk. Phosphatase, GGT 2. Used as Therapeutic agents. i.e Streptokinase( M.I), Serrato peptidase( anti-inflammatory) 3. Used as catalytic agents in laboratory. i.e Glucose Oxidase, Urease, Uricase (for respective blood tests)
- 52. Principal serum enzymes used in clinical diagnosis Serum Enzyme Major Diagnostic Use Aminotransferses Aspartate aminotransferase (AST or SGOT) Alanin aminotransferase (ALT, or SGPT) Myocardial infarction Viral Hepatitis Amylase Acute pancreatitis Ceruloplasmin Hepatolenticular degeneration (Wilson’s disease) Creatine kinase Muscle disorder & myocardial infarction γ – Glutamyl transpeptidase Various liver diseases (alcoholism) Lactate dehydrogenase (isozyme) Myocardial infarction Lipase (urinary) Acute pancreatitis Phosphatase, acid Metastatic carcinoma of the prostate Phosphatase, alkaline Various bone disorders, obstructive liver diseases
- 53. Important Question Short notes: Coenzymes, cofactors, isoenzymes, Km, allosteric enzymes, enzyme specificity, enzyme inhibition, active site, turnover rate. SEQs: 1. Define enzyme & classify them with example. 2. Discuss the mechanism of enzyme action. 3. Describe the various factors affecting enzyme activity. 4. Enlist the important serum enzymes of clinical interest. 5. Give at least three important uses/applications of enzymes. 6. Enumerate four important properties of enzyme. 7. Write down the MM equation & lineweaver-Burk equation. 8. Enlist the cardiac & liver disease diagnostic enzymes.
- 54. MCQs Enzymes markedly elevated in obstructive jaundice : i. GGT ii. 5-Nucleotidase iii. Alk.phosphatase iv. All of them Enzyme used as medicine in bleeding disorders : i. Streptokinase ii Thrombine iii.Kallekarein iv.Pepsin v. Hyaluronidase Enzymes increase the rates of reaction by : a) Increasing free energy of activation b) Decreasing free energy change of reaction c) Decreasing the energy of activation. d) Changing equilibrium constant of reaction An allosteric enzyme influences the enzyme activity by : i. Changing enzyme conformation by binding to site other than active site. ii. Competing for the catalytic site with the substrate iii. Changing the specificity of enzyme for the substrate iv. Changing the nature of the products formed A competitive inhibitor of enzyme has which of the following property ? i. It is frequently a feedback inhibitor ii. It becomes covalently attached to enzyme iii. It causes irreversible inactivation of enzyme iv. It interferes with substrate binding to enzyme.
- 55. Y-Glutamyl transpeptidase activity in serum is elevated in (a) Pancreatitis (b) Muscular dystrophy (c) Myocardial infarction (d) Alcoholism. In recent years, a non-protein compound has been identified to bring about catalysis in biological system. The name of the compound is (a) DNA (b) RNA (c) Lipids (d) Carbohydrates. Methotraxate is a anticancer drug. It competitively inhibit folic acid. Which of the following enzyme inhibited by this drug? a) Dihdropteroate synthesis b) Dihydro folate reductase c) Xanthine oxidase d) Lactate dehydrogenase e) Succinate dehydrogenase In obstructive liver disease, which of the following enzyme is elevated ? a) Acid phosphatase b) Alkaline phosphatase c) Amylase d) AST e) ALT Which of the following isoenzyme of lactate dehydrogenase is elevated in Myocardial infarction? a) LDH1 b) LDH2 c) LDH3 d) LDH4 e) LDH5 Allopurinol is drug used against gout which of following enzyme inhibited by this drug? a) Succinate dehydrogenase b) Xanthine oxidase c) Creatinine Kinase d) Kinas Thiamine pyrophosphate (T.P.P) is the coenzyme. It is derived from which of the following Vitamins? a) Vit. B1 b) Vit. B2 c) Vit. B6 d) Vit. B12 e) Biotin
- 56. Elevation of which of the following serum enzyme markers would be most useful in diagnosing a myocardial infarction in a patient who comes to your office 3 days after an episode of severe and prolonged substernal chest pain? a. LDH isoenzymes b. CKMB c. Troponin I d. Myoglobin e. Enzyme markers are no longer useful 3 days after an MI When [S] is equal to Km, which of the following condition exists ? a) Half of the enzyme molecules are bound to substrate. b) The velocity of the reaction is equal to Vmax c) Enzyme is completely saturated with substrate d) The reaction has reached the equilibrium "Lock& Key" model enzyme action proposed by Fischer implies that: a) The active site is flexible& adjusts to substrate b) The active site requires removal of PO4 c) The active site is complementary in shape to substrate. d) All of them v. None of them In competitive inhibition of enzyme action : i. Apparent Km is decreased ii. Apparent Km is increased. iii. Vmax is increased iv. Vmax is decreased A person aged 48 years presented in CCU with history of retrosternal chest pain radiating to left arm, breathlessness & sweating since 18 hours. a) What is your possible diagnosis? b) What enzymes would be raised in this patient? c) Why are enzymes raised in certain functional diseases? d) What other investigations will you suggest? e) Along with treatment what will you advise this patient for future care?
- 57. Thank You