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Enzymes by Dr. Aritri Bir

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Undergraduate Enzymology for MBBS students

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Enzymes by Dr. Aritri Bir

  1. 1. ENZYMOLOGY Dr. Aritri Bir Associate Professor Dept. of Biochemistry IQ City Medical College, Durgapur.
  2. 2. Enzymology (part I) • What is ENZYME ? • Nomenclature of ENZYMES. • Classifications of ENZYMES. • Structural organization of ENZYME. • What are Holoenzyme, Apoenzyme, Prosthetic group, Metallozyme, Metal dependent enzyme, coenzyme ? • Mechanism of enzyme action. • Factors affecting enzyme kinetics.
  3. 3. Introduction  AII enzymes are proteins except – RNA molecules  Organization of Enzyme molecule :  Monomeric and multimeric enzymes  Multifunctional enzyme  Multienzyme complex  The catalytic activity of enzyme depends on the integrity of its native conformation. - Explain  The catalytic activity of enzyme is usually lost if  If an enzyme is denatured or dissociated into its subunits  If an enzyme is broken down into its component amino acids  Thus the primary, secondary, tertiary, and quaternary structures of protein enzymes are essential to their catalytic activity.
  4. 4. Introduction - terminologies • Proenzyme / Zymogens : • Cofactors – Additional non-protein molecule essential for catalytic activity. • Two types : 1. Metal ions 2. Coenzymes • Coenzymes : Non-protein organic molecules. • Prosthetic groups : conenz or cofactors bound covalently to enz. • Apoenzymes & Holoenzymes :
  5. 5. Metal Activated Enzymes versus Metalloenzymes Metal-activated enzymes : shows greater catalytic activity by the presence of a mono or divalent metal ion exterior to the protein (in the assay medium). • Metal-activated enzymes typically lose activity during purification. • Hexokinase requires Mg2+ Metalloenzymes - metal as cofactor bound firmly to a specific region on the protein surface. • Metal ions cannot be separated without molecular breakdown of enzyme. • Cytochrome oxidase contains Fe3+ and Cu2+ • Catalase contains Fe2+
  6. 6. IUBMB Classification Enzymes are classified according to the type of reaction they catalyze. Cl. no Class Name Reaction they catalyze 1 OXIDOREDUCTASE Transfer of electrons (Oxidation & Reduction) 2 TRANSFERASE Transfer of groups 3 HYDROLASE Hydrolysis (cleavage of substrate by adding water) 4 LYASE Addition of groups to double bonds, or formation of double bonds by removal of group 5 ISOMERASE Transfer of groups within molecules to yield isomeric forms 6 LIGASE Formation of C-C, C-S, C-O, AND C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor
  7. 7. Nomenclature of ENZYMES • Hexokinase – IUBMB no. 2. 7. 1. 1. Class 2 Subclass 7 – transfers phosphate group 1st sub-subclass in which the acceptor of phosphate group is alcohol First enzyme of the 1st sub-subclass
  8. 8. ENZYME KINETICS • Study of the rate of enzyme-catalyzed reaction and the factors affecting it. • Kinetic theory of any chemical reaction ??? • Energy Barrier & Activation energy • Transition state • How the enzymes work?? • ES complex formation (reversible) by weak non-covalent interaction. • Each interaction release small amount of free energy • Binding energy released used to lower the activation energy • Reduces entropy • Desolvation of substrate • Proper alignment of functional group
  9. 9. FACTORS affecting THE RATE OF ENZYME CATALYSED REACTION • Temperature Each enzyme works in an optimal (narrow range) temperature – explain. • Initial temp increase → Activity increases gradually • At optimal temp → Activity maximum • Further increase in temp → Activity lost • pH • Enzyme concentration • Substrate concentration
  10. 10. Enzymology (part II) 1. Measurement of Enzyme activity. 2. Enzyme specificity 3. Michaelis Menten EQUATION. 4. Exception of MM Equation 5. Define Km and Vmax. What is its importance? 6. Determination of Km and Vmax by double reciprocal plot. 7. Turnover number of enzyme. 8. What are the different mechanism of enzyme catalysis?
  11. 11. Measurement of Enzyme Activity • Enzymes are quantitatively expressed in terms of catalytic activity. • The catalytic activity of an enzyme is indicated by the conversion of substrate into product. • International Unit: Amount of enzyme catalyzing the transformation of one micromole of substrate into product in one minute in 250 C. • KATAL: The amount of enzyme which converts 1 mole of substrate into product in 1 second. 1 Katal = 6 x 107 IU
  12. 12. Specificity of the enzyme The enzyme specificity is of Three different types: 1. Optical specificity – for substrate and Product. 2. Reaction Specificity – one enzyme catalyzes only one of the various reactions. 3. Substrate specificity a. Absolute b. Relative i. Group dependent ii. Bond dependent
  13. 13. Michaelis Menten Equation • The relation between Substrate conc and Reaction velocity is explained by the equation. Vo = Vo = initial rate of reaction Vmax = maximum rate of reaction Km = MM constant [S] = Substrate conc. Vmax [S] Km + [S]
  14. 14. Michaelis Menten Equation The prerequisite or assumptions: • Substrate concentration is much higher than the enzyme concentration. • The initial reaction velocity is considered. • The steady state hypothesis is followed. Exception: Allosteric enzymes
  15. 15. Km is Michaelis Menten constant. • It is the substrate concentration at which the reaction velocity is half of the maximum reaction velocity. • Significance: • It is one unique property of enzyme and constant for a specific substrate. • It indicates substrate affinity. • Low Km – higher affinity • High Km – lower affinity
  16. 16. Turnover Number of Enzyme • The number of substrate molecules converted to product in a unit time by one single molecule of enzyme when it is fully saturated. • Under standard condition, Vmax is equal to its turnover number. • Enzymes with higher turnover numbers are more efficient.
  17. 17. Lineweaver Burk Plot • Also known as Double Reciprocal Plot. Advantages over MM plot: • Determination of Km and Vmax is accurate. • Explanation of different enzyme inhibition is possible.
  18. 18. Mechanism of Enzyme catalysis • The enzyme catalysis and its specificity are best explained by ACTIVE SITE or The CATALYTIC SITE. • Extremely specific for substrate binding • Complex three dimensional form and shape • Nonpolar crevice The Enzyme substrate complex formation are of two types: 1. Fischer’s Lock and Key Model 2. Koshland’s Induced Fit Model.
  19. 19. Fischer’s Lock and Key Model • The active site is a rigid preshaped template. • Already in proper conformation even in absence of substrate. • Substrate fits in the active site of the enzyme just like a key fits in a lock (structural complementarity). • This model cannot explain the allosteric modulators
  20. 20. Koshland’s Induced Fit Model • Here, the active site conformation is flexible and dynamic. • Substrate binds to the enzyme to form ES complex intermediate. • Substrate binding to the enzyme induces conformational change in the active site to attain the final catalytic shape. • The enzyme in turn induces a reciprocal change in substrate to alter the configuration to transition state.
  21. 21. Enzymology (part III) • What are the different types of Enzyme inhibition? • What is Competitive Enzyme Inhibition? What are the kinetic changes? Give examples. Illustrate with Double reciprocal plot. • What is Non-competitive Enzyme Inhibition? What are the kinetic changes? Illustrate in Double reciprocal plot. • What is Irreversible Enzyme Inhibition? What are the kinetic changes? Give examples. Illustrate in Double reciprocal plot. • What is suicidal enzyme inhibition? Give examples.
  22. 22. Types of Enzyme Inhibition Reversible Irreversible Competitive Noncompetitive Uncompetitive
  23. 23. Reversible Competitive Inhibition • The inhibitor is structural analogue of the substrate. • It competes with the substrate for binding at the active site. Kinetic changes: By increasing the substrate concentration, inhibition can be reversed.
  24. 24. Reversible Competitive Inhibition • Vmax unchanged • Km increased.
  25. 25. Reversible Competitive Inhibition • Malonic Acid is the competitive inhibitor of Succinate Dehydrogenase. • Sulpher drugs contain sulphanilamide (structural analogue of PABA)- Inhibit the enzyme Dihydropteroate synthatase. • METHOTREXATE acts as anti-cancer drugs. • TRIMETHOPRIM acts as antibacterial drug. • PYRIMETHAMINE acts as antimalarial drug. • Dicumerol WARFARIN acts as anticoagulant drug.
  26. 26. Reversible Non-Competitive Inhibition • The inhibitor has no structural resemblance to substrate. • It binds to to the enzyme at different to active site – induces conformational changes in enzyme • Substrate binding occurs – but with decreased catalytic rate – product formation decreased. Kinetic changes: By increasing the substrate concentration, inhibition cannot be reversed.
  27. 27. Reversible Non-Competitive Inhibition • Vmax decreased • Km remains unchanged
  28. 28. Reversible Uncompetitive Inhibition • The inhibitor only binds to Enzyme-Substrate complex Kinetic changes: By increasing the substrate concentration, inhibition cannot be reversed. • Km and Vmax both decreased
  29. 29. • Inhibitors bind covalently to enzyme’s active site – active site modified and catalytic activity destroyed permanently. • Either functional group destruction or stable noncovalent adduct formation • Two types : oGroup specific reagents oAffinity labels Irreversible Inhibition 1. DIFP – Di isopropyl Fluro Phosphate 2. Iodoacetate TPCK – Chymotrypsin Inhibitor
  30. 30. • Here the inhibitor is the modified substrate. • Mechanism : • The inhibitor (substrate) binds an enzyme active site. • It is initially processed by the normal catalytic mechanism. • The mechanism of catalysis then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification. • Application: • In rational drug design – to optimize drug specificity and reduce side effects • Example : Allopurinol Aspirin, Ornithine Decarboxylase, MAO inhibitors Suicide Inhibition / Mechanism based Inhibition
  31. 31. 1. What are the different mechanisms to regulate enzyme activity? 2. What is allosteric regulation of enzyme activity? Illustrate with examples and graphical representation. 3. What are the different covalent modification which regulate enzyme activity? 4. What is long term regulation of enzyme activity? 5. What is feedback Inhibition? Give examples. Enzymology (part IV)
  32. 32. Regulation of Enzyme Activity Regulation through conformational changes Regulation through changes in amount of enzyme Regulation of metabolic pathways By Allosteric enzyme By Covalent Modification By Protein-Protein interaction Proteolytic Cleavage Regulated Enzyme Synthesis Regulated Protein Degradation Regulation of Rate Limiting Step Feedback Inhibition Tissue isoenzymes of regulatory enzymes Compartmentation Counter regulatory pathways
  33. 33. Allosteric Regulation of Enzyme Activity • Allosteric activators and inhibitors (allosteric effectors) are compounds that bind to the allosteric site (a site separate from the catalytic site) • It cause conformational change that affects the affinity of the enzyme for the substrate. COOPERATIVITY IN SUBSTRATE BINDING TO ALLOSTERIC ENZYMES • Usually an allosteric enzyme has multiple interacting subunits • They exist in active and inactive conformations • The allosteric effector promotes or hinders conversion from one conformation to another. • The binding of substrate to one subunit facilitates the binding of substrate to another subunit. • The first substrate molecule has difficulty in binding to the enzyme because all of the subunits are in the conformation with a low affinity for substrate (the taut “T” conformation) • The first substrate molecule to bind changes its own subunit and at least one adjacent subunit to the high-affinity conformation (the relaxed “R” state.) • The change in one subunit facilitated changes in all four subunits, and the molecule generally changed to the new conformation in a concerted fashion.
  34. 34. Allosteric Regulation of Enzyme Activity
  35. 35. ALLOSTERIC ACTIVATORS AND INHIBITORS • Activators bind at the allosteric site, a site physically separate from the catalytic site. The binding changes the conformation of the catalytic site in a way that increases the affinity of the enzyme for the substrate. • Activators bind more tightly to the high-affinity R state of the enzyme than the T state (i.e., the allosteric site is open only in the R enzyme) Thus, the activators increase the amount of enzyme in the active state, thereby facilitating substrate binding in their own and other subunits. • In contrast, allosteric inhibitors bind more tightly to the T state. Allosteric Regulation of Enzyme Activity
  36. 36. Allosteric Regulation of Enzyme Activity
  37. 37. Allosteric Regulation of Enzyme Activity
  38. 38. Covalent Modification PHOSPHORYLATION • Phosphorylation by a protein kinase or dephosphorylation by a protein phosphatase. • Phosphate is a bulky, negatively charged residue that interacts with other nearby amino acid residues - conformational change in active site. • The conformational change makes certain enzymes more active and other enzymes less active. • The effect is reversed by a specific protein phosphatase that removes the phosphate by hydrolysis. • Acetylation • ADP Ribosylation
  39. 39. Covalent Modification by Phosphorylation
  40. 40. Protein – Protein Interaction • Calcium – Calmodulin family of Modulator protein • Small G protein
  41. 41. Proteolytic Cleavage • Many proteolytic enzymes are secreted as Proenzymes, which are precursor proteins. • They undergo proteolytic cleavage to become fully functional (conformational changes to expose the active site). • Chymotrypsinogen is stored in vesicles within pancreatic cells until secreted into ducts leading to the intestinal lumen. In the digestive tract, chymotrypsinogen is converted to chymotrypsin by the proteolytic enzyme trypsin, which cleaves off a small peptide from the N-terminal region (and two internal peptides). • As the process is irreversible, specific inhibitor is required to inactivate it.
  42. 42. Regulation through changes in amount of enzyme Protein synthesis begins with the process of gene transcription. The code in messenger RNA is then translated into the primary amino acid sequence of the protein. • Generally the rate of enzyme synthesis is regulated by increasing or decreasing the rate of gene transcription, processes generally referred to as induction (increase) and repression (decrease). The content of an enzyme in the cell can be altered through selective regulated degradation as well as through regulated synthesis. • All proteins in the cell can be degraded with a characteristic half-life • Protein degradation via two specialized systems, Proteosomes and Caspases
  43. 43. Regulation of Enzyme Activity Regulation through conformational changes Regulation through changes in amount of enzyme Regulation of metabolic pathways Regulation of Rate Limiting Step Feedback Inhibition Tissue isoenzymes of regulatory enzymes Compartmentation Counter regulatory pathways Regulated Enzyme Synthesis Regulated Protein Degradation By Allosteric enzyme By Covalent Modification By Protein-Protein interaction Proteolytic Cleavage
  44. 44. Enzymes : Its Clinical Importance • Plasma Functional Enzymes: • Actively secreted in the blood in appropriate amount for their desired function in body. • Eg – Blood coagulation enzymes. • Plasma Nonfunctional Enzymes: • These enzymes have no definitive function in body. They are only released from their location during normal wear and tear. • They are commonly used in diagnostic purposes.
  45. 45. ISOENZYMES • Physically distinct molecular form of same enzyme with same catalytic activity but with different physical and chemical properties. • Oligomeric with multiple subunits - Homomultimeric & Heteromultimeric. • Only oligomeric form is active. • Properties : oOrigin oDistribution oTemperature stability oElectrophoretic mobility oKm values for substrate oInhibitor sensitivity • Importance: • Separation of Isoenzymes
  46. 46. Lactate Dehydrogenase • Five isoenzymes with different subunit composition (tetramer ) • Two types of subunit – H and M – encoded by different gene • Function – catalyzes interconversion of pyruvate and lactate using NADH/NAD+ as coenzyme.
  47. 47. Creatine Phosphokinase • Three Isoenzymes (Dimer) • Two Subunits : B & M • Function : Formation of creatine phosphate from creatine using ATP.
  48. 48. • LDH • CPK • Transaminases • Alkaline Phosphatase • Acid Phosphatase • Amylase and Lipase • Streptokinase • Pepsin and Trypsin Restriction Endonuclease • Glucose Oxidase • Peroxidase Enzymes : Its Clinical Importance Therapeutic purposesDiagnostic purposes Used in Laboratory as reagent Research purposes
  49. 49. 1. Lineweaver Burk Plot is commonly known as a) Double Logarithmic plot b) Double Parabolic plot c) Double Reciprocal plot d) Double Binomial plot
  50. 50. 2. Michaelis Menten Equation is Vo + [S] Km [S] c. Vmax = Vmax [S] Km + [S] a. Vo = Vmax [S] Km + [S] b. Vo = Km [S] Vo + [S] d. Vmax =
  51. 51. 3. Induced Fit Model was proposed by a) Fischer b) Koshland c) Michaelis d) Einstein
  52. 52. 4. Dicoumarol is structural analogue of a) Folic Acid b) Vitamin K c) PABA d) Succinic Acid
  53. 53. 5. Allopurinol inhibits the enzyme Xanthine Oxidase. This is a type of a) Competitive inhibition b) Noncompetitive Inhibition c) Uncompetitive Inhibition d) Irreversible Inhibition
  54. 54. 6. The enzyme Phosphoglucomutase falls under the class a) Class 4 b) Class 3 c) Class 5 d) Class 6
  55. 55. 7. International Unit of enzyme activity is a) Amount of enzyme catalyzing the transformation of one micromole of substrate into product in one minute. b) Amount of enzyme catalyzing the transformation of one mole of substrate into product in one minute. c) Amount of enzyme catalyzing the transformation of one micromole of substrate into product in one second. d) Amount of enzyme catalyzing the transformation of one mole of substrate into product in one second.
  56. 56. 8. Organophosphate compound inhibits the Acetylcholinesterase by a) Competitive inhibition b) Noncompetitive Inhibition c) Uncompetitive Inhibition d) Irreversible Inhibition
  57. 57. 9. What type of enzyme inhibition is represented in the double reciprocal plot ? a) Competitive inhibition b) Noncompetitive Inhibition c) Uncompetitive Inhibition d) Irreversible Inhibition
  58. 58. 10. One of the assumption based on which Michaelis Menten Equation is formulated is a) Km = [S] when V0 = Vmax b) Rates of formation and breakdown of Enzyme-substrate complex are equal c) In competitive enzyme inhibition, Vmax remaiins unchanged. Vmax [S] Km + [S] d) Vo =
  59. 59. 11. Catalase is an example of a) Coenzyme b) Proenzyme (Zymogen) c) Metallozyme d) Non-functional enzyme
  60. 60. 12. The relation between Katal and SI unit of enzyme activity is a) 1 Katal = 6 x 107 IU b) 1 Katal = 7 x 106 IU c) 1 IU = 6 x 107 Katal d) 1 IU = 7 x 106 Katal
  61. 61. 13. Turnover Number of Enzyme is a) The number of substrate molecules converted to product in a unit time by one mole of enzyme when it is fully saturated. b) The number of enzyme molecules required to convert 1 mole of substrate to product in a unit time when enzyme is fully saturated. c) The number of substrate molecules converted to product in a unit time by one single molecule of enzyme when it is fully saturated. d) The number of enzyme molecules required to convert 1 micromole of substrate to product in a unit time when enzyme is fully saturated.
  62. 62. 14. Enzyme a) Increases activation energey b) Decreases activation energy c) Increases free energy d) Decreases free energy
  63. 63. 15. If temperature is increased beyond optimal temperature, the enzyme activity is decreased because a) The substrate is dissociated from the enzyme b) The enzyme is denatured c) The product causes feedback inhibition d) The substrate is melted.
  64. 64. 16. All are enzyme kinetic plots except a) Eadie Hofstee Plot b) Michaelis Menten Plot c) Lineweaver Burk Plot d) Ramachandran Plot
  65. 65. 17. In competitive inhibition a) Vmax unchanged, Km decreases b) Vmax decreases, Km decreases c) Vmax decreases, Km increases d) Vmax increases, Km unchanged
  66. 66. 18. Allosteric activators a) Induces the stability of ‘T’ form of enzyme b) Binds to the active site of the enzyme c) Re-orient the alignment of amino acids surrounding the active site d) Denatures the enzyme
  67. 67. 19. Advantage of Lineweaver Burk plot over MM plot is a) Allosteric regulation of enzymes can be studied b) Estimation of Km and Vmax is more accurate c) Initial and terminal velocity – both can be considered d) Pre-steady state kinetics can be studied more precisely
  68. 68. 20. Activation and deactivation of enzyme by Protein Kinase and Phosphatase is a type of modification of a) Electrostatic bond b) Covalent bond c) Hydrogen bond d) James Bond

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