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Enzymalogy Factors affecting enzyme activity and kinetics

A comprehensive presentation on Factors affecting enzyme activity & Kinetics of Enzymes for MBBS ,BDS, B Pharm & Biotechnology students to facilitate self- study.

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Enzymalogy Factors affecting enzyme activity and kinetics

  1. 1. Enzymology: Factors affecting enzyme activity& Kinetics Dr. Rohini C Sane
  2. 2. Factors affecting enzyme activity 1. Concentration of enzymes 2. Concentration of substrate 3. Concentration of products 4. Temperature 5. p H 6. Activators (Coenzymes & Cofactors ) 7. Inhibitors 8. Time 9. UV light & Radiations 10. Oxidizing agent 11. Anti-enzymes
  3. 3. Factors affecting enzyme activity: concentration of enzymes Rate of reaction directly proportional to concentration of enzymes (when p H ,temp ,substrate concentration etc are constant ) Clinical application 1. Determination of enzyme concentration 2. For diagnosis of inborn errors in metabolism
  4. 4. Factors affecting enzyme activity: Concentration of Product Factors affecting enzyme activity: Concentration of Product 1. E+ S P+ E 2. With increase in product concentration velocity of reaction decreases 3. Feed back mechanism 4. Inborn errors of metabolism: there is increase in substrate concentration in plasma & product concentration decreases 5. eg Phenylketonuria ( Phenylalanine  Tyrosine- catalyzed by Phenylalanine Hydroxylase. Enzyme is inhibited /absent concentration of Serum Phenylalanine increases & concentration Serum Tyrosine decreases ) A-- - B
  5. 5. Factors affecting enzyme activity: concentration of product
  6. 6. Factors affecting enzyme activity: Concentration of products Increase in Concentration of products ( after saturation of enzyme molecules ) decrease in velocity of reaction  Feed Back Inhibition
  7. 7. Factors affecting enzyme activity: concentration of Substrate • 8 E + 2S ↔ 2 ES  2P + 8E ( 1HR ) • 8 E + 4S ↔ 4 ES  4P + 8E ( 1/2HR ) • 8 E + 8S ↔ 8 ES  8P + 8E ( 1/4HR ) SATURATION--------------------------------------------------------------- • 8 E + 10S ↔ 8 ES  8P + 8E ( 1/4HR ) • 8 E + 12S ↔ 8 ES  8P + 8E ( 1/4HR ) • *( Rate of formation of 2P )
  8. 8. (S ) –Substrate Concentration V ₒ -- Initial Velocity K m -- Michelis Menten constant Vmax –maximum velocity Factors affecting enzyme activity: concentration of Substrate
  9. 9. Line Weavers Burk Equation-Double Reciprocal Graph 1/VO =Km /Vmax (1/S ) + 1/Vmax Y = mx + c ( slope = Km /Vmax ) When 1/S =0 S=∞ 1/Vₒ = 1/V max Advantage : (exact Vmax )
  10. 10. Michaelis Menten equation • V = initial velocity • Vmax = maximum velocity • Km = Michaelis–Menten constant • S = substrate concentration Importance of Michaelis Menten equation : 1. relates substrate concentration with reaction velocity 2. Quantitative calculations of enzyme characterization 3. Analysis of enzyme inhibition The plot provides a useful graphical method for analysis othe Michaelis–Menten equation:
  11. 11. Enzyme Kinetics Increase in rate of reaction is observed when : (1)increasing internal energy ( activation energy) by increase in temperature  increase in molecules in transition state (2) cell when uses of enzyme ,there is a) Decrease in activation energy b) Increase in number of substrate in transition state c) Increase in rate of reaction
  12. 12. Rate of enhancement by Enzymes • Chemical reaction Substrate transition state product Number of collision α rate of reaction Transition state : reactive form Activation energy : of the reaction is the amount energy in calories required to bring all molecules in one molar of substrate at a given temperature to transition state (at the top of energy barrier ).
  13. 13. Rate enhancement by enzyme in catalyzed reaction Number of substrate molecule Internal energy ( kcal ) 20 * √ 150 50 √ 60 30 10 Activation energy (Uncatalyzed reaction) 130 Transition state 130 Number of substrate molecules converted into product Catalyzed reaction Activation energy Number of molecules in transition state = number of substrate molecules into product 20* 50 70 √
  14. 14. Energy diagram for chemical reaction
  15. 15. Activation energy for Catalyzed & uncatalyzed reaction
  16. 16. Factors affecting enzyme activity: Temperature Each Enzyme has optimum temperature Q 10 = 10 ⁰C reaction rate doubled HIGHER TEMPERATURE CAUSES DENATURATION OPTIMUM TEMPERATURE FOR MOST BODY ENZYMES = 37 ⁰C OPTIMUM TEMPERATURE FOR UREASE =60 ⁰C
  17. 17. Factors affecting enzyme activity: p H Optimum p H : Pepsin -1-2 ,Glucose 6-phosphtase -7.2 ,Alkaline Phosphatase- 11
  18. 18. Factors affecting enzyme activity: p H Optimum p H :Pepsin -1-2 ,Urease -6.5 ,Trypsin-7.5
  19. 19. Factors affecting enzyme activity : presence of Oxidizing agent Oxidation by oxygen or by oxidizing agents E SH + ½ O2 E S + H2O SH S E S + 2 R-SH ↔ E SH + R –S-SH S SH Reduced Sulph-hydryl group is contributed by Cysteine or Glutathione
  20. 20. Factors affecting enzyme activity: Radiation X rays ,Beta or Gamma rays ---( high energy rays ) peroxides + E ↓ OXIDIZED ENZYMES ↓ LOSS OF ENZYME ACTIVITY ↑ LOSS OF GENE EXPRESSION
  21. 21. Factors affecting enzyme activity: Anti –enzymes • Serum containing Anti enzymes /Antibodies against enzymes eg Anti -Trypsin, Anti –Pepsin  decreased /loss of activity
  22. 22. Use of Anti enzyme in treatment of Myasthenia Gravis
  23. 23. Definition of Cofactors & Coenzymes Factors affecting enzyme activity: Co-enzyme & Cofactors (Activators )
  24. 24. Factors affecting enzyme activity: co-enzyme & cofactors
  25. 25. Comparison of Cofactors & Coenzymes
  26. 26. Coenzyme forms of Vitamin B & their functions Vitamin Activated form- (coenzyme ) Type of catalysis Enzyme using co - enzyme Thiamine Thiamine Pyrophosphate(TPP ) Aldehyde or Keto Group Trans- Ketolase Riboflavin Flavin Mono Nucleotide (FMN ) Hydrogen or Electron L -Amino oxidases Riboflavin Flavin Adenine Dinucleotide (FAD ) Hydrogen or Electron D -Amino oxidases Niacin Nicotinamide Adenine Dinucleotide (NAD ) Hydrogen or Electron LDH Niacin Nicotinamide Adenine Dinucleotide Phosphate (NADP ) Hydrogen Or Electron G-6 P-D Lipoic Acid Lipoic Acid Hydrogen Or Electron Pyruvate Dehydrogenase Complex
  27. 27. Coenzyme forms of Vitamin B & their functions Vitamin Activated form- (coenzyme ) Type of catalysis Enzyme using coenzyme Pyridoxine Pyridoxal Phosphate Amino Group Transfer Alanine Transaminase Pantothenic Acid Coenzyme A Acyl Group Transfer Thio Ketolase Folic Acid Tetra Hydro Folate (TFH4 ) One Group Transfer- formyl, Methyl Formyl Transferase Biotin Biotin CO2 Pyruvate Carboxylase Cobalamine Methyl Cobalamine Methyl Malonyl Co A Mutase
  28. 28. VITAMINS AND COENZYMES Vitamin Coenzyme Reaction type Coenzyme class SOURCE: Compiled from data contained in Horton, H. R., et al. (2002). Principles of Biochemistry , 3rd edition. Upper Saddle River, NJ: Prentice Hall. B 1 (Thiamine) TPP Oxidative decarboxylation Prosthetic group B 2 (Riboflavin) FAD Oxidation/Reduction Prosthetic group B 3 (Pantothenate) CoA - Coenzyme A Acyl group transfer Cosubstrate B 6 (Pyridoxine) PLP Transfer of groups to and from amino acids Prosthetic group B 12 (Cobalamin) 5-deoxyadenosyl cobalamin Intramolecular rearrangements Prosthetic group Niacin NAD + Oxidation/Reduction Cosubstrate Folic acid Tetrahydrofolate One carbon group transfer Prosthetic group Biotin Biotin Carboxylation Prosthetic group Read more:
  29. 29. Factors affecting enzyme activity: Cofactors (activators ) Cofactor –inorganic ion Enzymes Fe 2 ⁺ ,Fe3 ⁺ Peroxidase Cu ⁺ ⁺ Cytochrome oxidase Mg ⁺⁺ Hexokinase Ni⁺⁺ Urease Mn ⁺⁺ Arginase K ⁺ Pyruvate Kinase Zn ⁺ ⁺ DNA Polymerase Mo⁺⁺ Nitrate Reductase Se Glutathione Peroxidase Ca ⁺ ⁺ Lipase Cl⁻ Salivary Amylase
  30. 30. Factors contributing catalytic efficiency 1. Proximity & orientation of substrate in relation to catalytic group 2. Strain & orientation of the susceptible bond by induced fit of enzymes 3. General acid base catalysis 4. Covalent catalysis
  31. 31. Effects of proximity & orientation- enhancement of catalytic efficiency of enzymes
  32. 32. Effects of proximity & orientation-increased catalytic efficiency of enzymes
  33. 33. Proximity & Orientation of substrate in relation to catalytic group • Orientation : unfavorable • Proximity : unfavorable no product formation • Orientation : unfavorable • Proximity : favorable no product formation • Orientation :favorable • Proximity : favorable product formation ES has lower activation energy as ES IN TRANSITION STATE
  34. 34. Induced fit hypothesis Of enzyme catalysis – Transition state stabilization leads to rate enhancement
  35. 35. Induced fit model of enzyme catalysis: change in three dimensional structure of enzyme & substrate is induced on binding of substrate to enzyme active site
  36. 36. Induced Fit ,Strain & Distortion Relaxed substrate molecule +Relaxed enzyme conformational change  strain form of substrate molecule 1. Strain active site 2. Distortion of substrate 3. Conformational leverage on substrate • NESSECIATES: ENZYME LARGE & PROTEIN MOLECULE
  40. 40. Acid Base catalysis Proton is transferred From amino acid of enzyme to substrate This results in lowering activation energy or stabilization in transition state.
  41. 41. Covalent Catalysis- Covalent linkage between amino acid from active site of enzyme & substrate . This results in lowering activation energy or stabilization in transition state.
  42. 42. Covalent Catalysis- Products have lower affinity for enzyme active site & are therefore released. Enzymes are set free at end of reaction ( Completion of catalysis ) in unaffected form
  43. 43. Covalent intermediates of Enzymes in Covalent catalysis Class enzyme Serine Class Phospho Glucomutase Phospho Enzyme Serine Class Trypsin Acyl Enzyme Serine Class Chymotrypsin Acyl Enzyme Serine Class Acetyl Choline Esterase Acyl Enzyme Cysteine Class Pepsin Acyl Enzyme Cysteine Class Glyceraldehyde Phosphate Dehydrogenase Acyl Enzyme Histidine Class Glucose 6 Phosphatase Phospho Enzyme Histidine Class Succinyl –Coa Synthtase Phospho Enzyme Lysine Class Trans Aldolase Schiff Base Lysine Class Amino Acid Oxidase Schiff Base
  44. 44. Comparison between Acid- Base catalysis ,Covalent catalysis & Metal ion catalysis