Chapter 5 Enzymes What are enzymes? and How do they work?

1. Chapter 5 EnzymesWhat are enzymes?and How do they work?

2. 5.1 Introduction to Enzymes Enzymes are catalysts What properties would an ideal catalyst have?

3. 5.1 Introduction to Enzymes What properties would ideal catalysts have? • High degree of specificity for their substrates. • Accelerate chemical reactions tremendously. • Function in mild conditions.

4. 5.1 Introduction to Enzymes

5. 5.1 Introduction to Enzymes

6. 5.1 Introduction to Enzymes

7. 5.1 Introduction to Enzymes

8. 5.1 Introduction to Enzymes

9. 5.1 Introduction to Enzymes A Few Definitions Cofactor, Coenzyme, Prosthetic groupHoloenzymeApoenzyme

10. 5.1 Introduction to Enzymes Cofactor – additional chemical component needed for catalysis. - often an inorganic metal ion (mineral).

11. 5.1 Introduction to Enzymes Coenzyme – complex organic molecule needed for catalysis. - often a vitamin

12. 5.1 Introduction to Enzymes Prosthetic group – non amino acid portion of the enzyme needed for catalysis. Often a coenzyme or metal ion. Holoenzyme – complete catalytically active enzyme, with all necessary prosthetic groups. Apoenzyme – The protein part of the holoenzyme. Prosthetic groups are absent.

13. 5.1 Introduction to Enzymes

14. 5.1 Introduction to Enzymes Classification of Enzymes

15. 5.1 Introduction to Enzymes Classification of Enzymes

16. 5.2 How Enzymes work Consider the conversion of S  P (uncatalyzed reaction)

17. 5.2 How Enzymes work E + S  ES  EP  E + P

18. 5.2 How Enzymes work E + S  ES  EP  E + P 1. Enzymes affect the reaction rate, not the equilibrium 2. Enzymes lower activation energy 3. Enzymes stabilize the transition state of a reaction

19. 5.2 How Enzymes work Three mechanisms of catalysis • General Acid-Base Catalysis • 2. Covalent catalysis • 3. Metal Ion Catalysis

20. (1) General Acid-Base Catalysis Charged intermediates formed during catalysis are stabilized by donation or acceptance of protons by an amino acid side chain in the active site

23. Amino acid side chains that can participate in acid-base catalysis

24. (2) Covalent catalysis A transient covalent bond is formed between enzyme and substrate during catalysis. After catalysis the covalent complex is released and free enzyme is regenerated. A-B + X:  A-X + B A + X: + B Substrate Amino Acid Covalent Products Amino Acid for catalysis side chain bond side chain

25. (2) Metal Ion Catalysis Metal ions bound to the enzyme participate in catalysis. The metal may form an ionic interaction with a substrate or mediate oxidation - reduction by donating or accepting electrons.

26. 5.3 Enzyme kinetic Initial rates of reaction are measured in enzyme kinetics E + S  ES  EP  E + P

27. The rate of reaction is dependent on substrate concentration [S] – substrate concentration Vo – initial velocity of a reaction. A significant amount of substrate has not yet been converted to product. Vmax – maximal velocity of a reaction. Addition of more substrate will not increase the rate of the reaction. Km – The concentration of substrate at which the rate of the reaction is half-maximal

28. Michaelis-Menten equation

30. The double-reciprocal plot Experimental determination of Vmax and Km

32. k1 k2 k3 E + S  ES  EP  E + P k-1 k-2 • k represents a rate constant. • The conversion of S to P is described by a series of rate constants. • The conversion of EP to E + P is often the rate-limiting step of the reaction. • k3 =kcat, turnover number. • kcat has the units of sec-1 (molecules / second)

33. Turnover numbers vary widely

34. Catalytic efficiencies of enzymes. kcat / Km = catalytic efficiency. The kcat / Km value is limited by the rate E and S can diffuse together in aqueous solution. The diffusion-controlled limit is 1 x 109 M-1sec-1

36. 5.4 Enzyme Substrate Interaction

37. 5.4 Enzyme substrate interaction • Catalytic site • Where the reaction actually occurs. • Binding Site • Area that holds substrate in proper place. • Enzymes uses weak, non-covalent interactions to hold the substrate in place based on R group of amino acids. • Shape is complementary to the substrate and determines the specificity of enzyme. • Sites are pockets or clefts on the enzyme surface

38. 5.4 Enzyme substrate interaction

39. 5.4 Enzyme substrate interaction

40. 5.4 Enzyme substrate interaction

41. 5.4 Enzyme substrate interaction

42. 5.5 Factors Affecting Enzyme Activity (1)

43. 5.5 Factors Affecting Enzyme Activity

44. 5.5 Factors Affecting Enzyme Activity

45. 5.5 Factors Affecting Enzyme Activity

46. 5.5 Factors Affecting Enzyme Activity (2)

47. 5.5 Factors Affecting Enzyme Activity

48. 5.5 Factors Affecting Enzyme Activity

49. 5.5 Factors Affecting Enzyme Activity (3) Enzyme Inhibition • Enzyme activity is decreased by inhibitors.This is the basis of many pharmaceutical agents. • Many substances can inhibit enzyme activity. • Inhibitors include substrate analogs, toxins, drugs, metal complexes.

50. 5.5 Factors Affecting Enzyme Activity • Two broad classes of inhibitors: • Irreversible and Reversible. • Irreversible: Forms covlent or very strong noncovalent bonds. The sites of attack is an amino acid group that participates in normal enzymatic reaction. • Reversible: Forms weak, noncovalent bonds that readily dissociate from an enzyme. The enzyme is only inactive when the inhibitor is present.