ENZYMES - Part 2

1. ENZYMES - Part 2

2. CONTENTS 1. Enzyme Inhibition 2. Regulation (control) of enzyme activity 3. Isoenzymes / Isozymes 4. Clinical applications of enzymes

3. ENZYME INHIBITION Definitions : Enzyme inhibitor : Is as a substance which binds to the enzyme, and brings about a decrease in the velocity of the enzyme-catalyzed reaction. Enzyme inhibition : Is the reduction of enzyme activity by binding of inhibitor to the enzyme.

4. ENZYME INHIBITION…….. Importance of enzyme inhibitors : • 1. Used to elucidate the mechanism of enzyme action. • Many drugs and poisons are enzyme inhibitors. E.g., : Group of Drugs : Anticancer, antibiotics, antivirals, immunosuppresents.

5. ENZYME INHIBITION…….. Features of an Inhibitor : • Enzyme • + • Inhibitor • Enzyme Inhibition 1. Where does the inhibitor bind on the enzyme? Inhibitor may bind (i) to the active site or (ii) to a site other than the active site. • it may be to a • Free enzyme molecule or • ES-complex.

6. Features of an Inhibitor……… (2.) What does it bind ? Binds to specific R-groups of amino acid residues, involved in , • catalysis, • substrate-binding or • maintenance of functional conformation of the enzyme.

7. Features of an Inhibitor……… (3.) What type of interactions between enzyme and inhibitor ? (i) Weak, non-covalent bonds - Reversible . • Ionic bond, • H-bond, • Hydrophobic bond. - irreversible . (ii) Strong covalent bond

8. Features of an Inhibitor……… (4.) What is the product of enzyme inhibitor interaction? Enzyme – Inhibitor complex is formed. i.e., EI complex

9. Types of Inhibition Enzyme inhibition 2 Types Basis : Stability of EI complex Reversible Irreversible • Weak non-covalent bonds • Easily dissociable • Enzyme activity is restored by removing the inhibitor . Competitive Non competitive Uncompetitive

10. I. Reversible inhibition: 3 types • Competitive • Non-competitive • Uncompetitive

11. Competitive inhibition Substrate Active site • Substrate • Complex Enzyme Enzyme Catalysis Structural analog Competitive Inhibitor No catalysis Products Enzyme - Inhibitor complex Enzyme • product • Complex Enzyme

12. Substrate Substrate Increase in [S] Active site S S S Enzyme Inhibition is reversed S Enzyme is active Competitive Inhibitor Catalysis Products Enzyme - Inhibitor complex No catalysis Enzyme • product • Complex ES complex EI complex Enzyme 1. Competitive inhibition

13. 1. Competitive inhibition Definition : Competitive inhibition is a type of • reversible inhibition in which • the inhibitor is a structural analog • of the substrate • and hence competes with the substrate for the active site.

14. Features of an competitive Inhibitor……… • At high [S], ‘S’ displaces the ‘I’ molecule • from the substrate binding site • and thus overcome the inhibition. Kinetics of an competitive Inhibition Kinetics - Effect on Vmax and Km

15. The Michelis-Menten plot - Competitive inhibition (Substrate-saturation curve). Vmax With inhibitor Km Km • Vmax remains unchanged. • Km increases.

16. Examples of Competitive Inhibition : Classic example: Malonate (structural analog of succinate). Succinate Fumerate Similarity : two carboxyl groups.

17. Therapeutic Applications of Competitive Inhibition:

18. Process of Non competitive inhibition Substrate Substrate ES Complex EP Complex Catalysis Active site Enzyme Inhibitor No Catalysis ESI Complex EI Complex

19. 2. Non competitive inhibition Definition : Uncompetitive inhibition is a type of • reversible inhibition in which • the inhibitor is NOT a structural analog • of the substrate • and hence DOES NOT compete with the substrate for the active site.

20. Features of an NON competitive Inhibitor……… • At high [S], ‘S’ cannot displace the ‘I’ molecule • from the substrate binding site • and thus cannot overcome the inhibition. Kinetics Kinetics - Effect on Vmax and Km

21. The Michelis-Menten plot – Non competitive inhibition (Substrate-saturation curve). Vmax With inhibitor Km Km • Vmax Decreases. • Km remains unchanged

22. Examples for NON competitive inhibitors • Trypsin inhibitors • occur in Soyabean and Ascaris parasites (worms). ii. -1 Antitrypsin which inhibits serine proteases (such as trypsin).

23. Process of Uncompetitive inhibition Substrate ES Complex EP Complex Catalysis Active site Enzyme Inhibitor No Catalysis ESI Complex

24. 2. Uncompetitive inhibition Definition : Uncompetitive inhibition is a type of • reversible inhibition in which • the inhibitor binds only to the ES complex • Increased [S], cannot overcome the inhibition.

25. The Michelis-Menten plot : Uncompetitive inhibition (Substrate-saturation curve). Vmax Without inhibitor Vmax. i With inhibitor • Vmax decreased. • Km decreased • Kinetics

26. Example for Uncompetitive Inhibition Phenylalanine uncompetitively inhibits enzyme placental alkaline phosphatase. Summary of effect of reversible inhibitors

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28. II. Irreversible inhibition: The inhibitor • Binds to the enzyme tightly • By covalent bonds • Forms a stable complex. • Enzyme-inhibitor complex does not dissociate significantly. Examples:A variety of poisons (toxic substances), oxidizing agents.

29. Examples for Irreversible inhibition A variety of poisons (toxic substances), oxidizing agents -irreversible inhibitors.

30. Examples for Irreversible inhibition………

31. Suicide inhibition / Substrate Substrate Active site Mechanism based inhibition Enzyme ES Complex Substrate analog No catalysis Inactive inhibitor Active inhibitor Catalysis Enzyme – Inhibitor complex Enzyme - Substrate analog complex Enzyme substrate analog complex Enzyme – Inhibitor complex

32. Suicide inhibition or Mechanism based inactivation: Is a special type of irreversible inhibition The inhibitor is a substrate analog is inactive initially binds to the active site of the enzyme. the Inhibitor is modified. Modified inhibitor is an effective inhibitor binds irreversibly and inhibits further reactions of the same enzyme .

33. Example of Suicide inhibition : i. Allopurinol HypoxanthinXanthin Uric acid Xanthin Oxidase Xanthin Oxidase ↑↑↑Uric acid leads to GOUT Structural analog Allopurinol Alloxanthin Xanthin Oxidase Clinical use of Allopurinol: Treatment of gout, for ↓↓ uric acid production. NOTE : cited as an example for competitive inhibition – NOT appropriate.

34. ii. 5 - Flurouracil Enzyme : Thymidine kinase Mechanism : “ Suicide inhibition”. required for synthesis of Thymidine triphosphate for synthesis of DNA. Application : used as an “ Anticancer drug”. iii. Fluroacetate : Enzyme : Aconitase (of TCA cycle).

35. Mechanisms in regulation of metabolic reactions 1. Regulation /control of enzyme activity 2. Membrane transport Major mechanism by which metabolism is controlled 3. Regulation by hormones and 4. Neuronal regulation. By Regulatory enzymes / key enzymes of metabolic pathways.

36. Regulatory enzymes / key enzymes Often these enzymes • Catalyze the committed stepin a metabolic pathway. • These reactions are irreversible. • These reactions are rate-limitingfor the whole pathway. • Regulatory enzymes are either • at or near the initial steps in a pathway. • Or part of a branch point or cross-over point between pathways.

37. Types of mechanisms for regulation of enzyme activities Mechanisms for regulation of enzyme activities (ii) Change of enzyme catalytic efficiency (i) Change of enzyme concentration Change in rate of enzyme degradation Change in rate of enzyme Synthesis ↓↓↓ Synthesis ↑↑↑ Synthesis Enzyme Repression Enzyme Induction

38. Types of mechanisms for regulation of enzyme activities (ii) Change of enzyme catalytic efficiency covalent Modification Non-covalent Modification (Reversible, Allosteric Enzymes) Irreversible Reversible Phosphorylation-Dephosphorylation LimitedProteolysis.

39. Types of mechanisms for regulation of enzyme activities Additional mechanisms • Multi-enzyme complex • Enzyme compartmentalization (i) CONTROL OF ENZYME SYNTHESIS: DNA  Inducers Hormones , other molecules ↑↑↑ ↓↓↓ Transcription  Repressors ↓↓↓ ↑↑↑ mRNA Enzyme Repression Enzyme Induction Translation ↓↓↓ ↑↑↑ Protein (Enzyme)

40. Definitions : Induction & Repression Induction : is ↑↑↑ synthesisof enzyme through ↑↑↑ gene transcription of the relevant mRNA, leading to an ↑↑↑ in the [E]. Inducible enzymes / Adaptive enzymes Repression : is an↓↓↓ synthesisof enzyme through ↓↓↓ gene transcription of the relevant mRNA, leading to a ↓↓↓ in the [E].

41. Examples of Adaptive enzymes

42. 2. CONTROL OF ENZYME DEGRADATION Site : Lysosomes / in complexes termed proteosomes. Regulation : involves transfer of a polypeptide, ubiquitin, to targeted enzymes (proteins).

43. Allosteric Enzymes and Allosteric regulation : Binds to Substrate Active site Catalysis Allosteric site Binds to Modifier / Effector ↓↓ activity ↑↑ activity Positive Modifier (+) Negative Modifier (-) Allosteric activator(+) Allosteric inhibitor(-)

44. Substrate Process of allosteric regulation S Allosteric enzyme Active site EP complex ES complex Enzyme Allosteric site is altered Active site is altered Allosteric site E Binding either S or E - confirmational change in the enzyme ↑↑ Effector E

45. 3. ALLOSTERIC REGULATION: Definition of an allosteric enzyme : is that enzyme whose • activity can be altered (modulated) • by binding of a certain molecule (effector/ modifier) • at a site other than the catalytic site • called allosteric site (allo=other, steros=space). Sites on an allosteric enzyme : • Catalytic site Substrate binding Modifier binding • Allosteric site

46. Many metabolic pathways are regulated by allosterric enzymes Substrate / starting material End Product Allosterric enzyme HOW ???? S A B C D E F P E1 E2E3 E4 E5 E6 E7 •  •  • ↑↑ • ↑↑ • starting material / an early intermediate • end product • feed forward activation • feedback inhibition

47. Examples for Allosteric inhibition.

48. 4. REGULATION BY COVALENT MODIFICATION: A. Reversible covalent modification: B. Irreversible covalent modification: Enzyme Active form Enzyme Inactive form Inter-convertible Addition – Removal of groups E.g., Phosphorylation – dephosphorylation Nucleotidation – denucleotidation (i.e., Adenylation / deadenylation)

49. REGULATION BY COVALENT MODIFICATION…… e.g.,Phosphorylation–dephosphorylation Protein Kinase ATP ADP Phosphorylated enzyme Enzyme De-phosphorylated enzyme Enzyme - P Ser, Thr, Tyr Pi H2O Phosphatase • Short-term • Readily reversible • Hormonal / other physiological signals.

50. REGULATION BY COVALENT MODIFICATION…… e.g.,Phosphorylation–dephosphorylation Some enzymes are active either in the phosphorylated form Or in the de-phosphorylated form.