HOW ENZYMES WORK

1. HOW ENZYMES WORK

2. Model of the surface of an enzyme. ENZYMES SPEED UP CHEMICAL REACTIONS Enzymes are biological catalysts – substances that speed a reaction without being altered in the reaction. Mostenzymes are proteins. Enzymes are essential for life.

3. Enzymes 􀁺Cofactors 􀁺 Coenzymes 􀁺 Holoenzyme 􀁺 Apoenzyme

4. How Enzymes Work? • Body conditions(temperature, pressure etc.) not good for reaction • Only enzymes can catalyse the reactions in this conditions • A special environment inside enzymes for reaction ACTIVE SITE • Molecule binds active site SUBSTRATE

8. Enzymes Lower a Reaction’s Activation Energy

9. Each reaction has a transition state where thesubstrate is in an unstable, short-livedchemical/structural state. Free Energy of Activation is symbolized by ΔG‡. Enzymes act by lowering the free energy of the transition state

10. Enzymes speed up metabolicreactions by lowering energy barriers • Enzyme speed reactions by lowering EA. – The transition state can be reached at moderate temperatures. • Enzymes do not change delta G. – It speed-up reactions that would occur eventually. • Because enzymes are so selective, they determine which chemical processes will occur at any time

11. Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate • The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction • Not a lock & key but induced fit – the enzyme and/or the substrate distort towards the transition state

13. Induced Fit • A change in the shape of an enzyme’s active site • Induced by the substrate

14. Lock and Key Model • An enzyme binds a substrate in a region called the active site • Only certain substrates can fit the active site • Amino acid R groups in the active site help substrate bind • Enzyme-substrate complex forms • Substrate reacts to form product • Product is released

16. Enzyme Kinetics - Kinetics The study of the rate of change. - Enzyme Kinetics Rate of chemical reactions mediated by enzymes. Enzymes can increase reaction rate by favoring or enabling a different reaction pathway with a lower activation energy, making it easier for the reaction to occur.

17. Michaelis-Menten kinetics Vmax approached asymptotically V0 varies with [S] V0 is moles of product formed per sec. when [P] is low (close to zero time) E + SESE + P Michaelis-Menten Model V0 = Vmax x[S]/([S] + Km) Michaelis-Menten Equation

18. Determining initial velocity (when [P] is low)

19. Steady-state & pre-steady-stateconditions At pre-steady-state, [P] is low (close to zerotime), hence, V0 for initial reaction velocity At equilibrium, no net change of [S] & [P] or of [ES] & [E] At pre-steady state, we can ignore the back reactions

20. Michaelis-Menten kinetics (summary) Enzyme kinetics (Michaelis-Menten Graph) : At fixed concentration of enzyme, V0 is almost linearly proportionalto [S] when [S] is small, but is nearly independent of [S] when [S]is large Proposed Model: E + S  ES  E + P ES complex is a necessary intermediate Objective: find an expression that relates rate of catalysis to the concentrations of S & E, and the rates of individual steps Start with: V0 = k2[ES],and derive, V0 = Vmax x[S]/([S] + Km) This equation accounts for graph data. At low [S] ([S] < Km), V0 = (Vmax/Km)[S] At high [S] ([S] > Km), V0 = Vmax When [S] = Km, V0 = Vmax/2. Thus, Km = substrate concentration at which the reaction rate (V0) is half max.

21. Range of Km values Km provides approximation of [S] in vivo for many enzymes

22. Lineweaver-Burk plot (double-reciprocal)

23. Eadie-Hofstee plot

24. Hanes-Woolf Plot

25. Allosteric enzymes • Allosteric enzymes tend to be multi-sub unit proteins • The reversible binding of an allosteric modulator (here a positive modulator M) affects the substrate binding site

27. Kinetics Models Cooperation vo (+) [S] (+) vo (+) [S] vo (-) (-) [S] Mechanism and Example of Allosteric Effect Allosteric site R = Relax (active) Homotropic (+) Concerted Allosteric site A Heterotropic (+) Sequential X Heterotropic (-) Concerted T = Tense (inactive) I X X

28. Enzyme Inhibitors • Specific enzyme inhibitors regulate enzyme activity and help us understand mechanism of enzyme action. (Denaturing agents are not inhibitors) • Irreversible inhibitors form covalent or very tight permanent bonds with aa at the active site of the enzyme and render it inactive. 3 classes: groupspecific reagents, substrate analogs, suicide inhibitors • Reversible inhibitors form an EI complex that can be dissociated back to enzyme and free inhibitor. 3 groups based on their mechanism of action: competitive, non-competitive and uncompetitive.

29. Enzyme Inhibition

30. Competitive inhibitors • Compete with substrate for binding to enzyme • E + S = ES or E + I = EI . Both S and I cannot bind enzyme at the same time • In presence of I, the equilibrium of E + S = ES is shifted to the left causing dissociation of ES. • This can be reversed / corrected by increasing [S] • Vmax is not changed, KM is increased by (1 + I/Ki) • Eg: AZT, antibacterial sulfonamides, the anticancer agent methotrexate etc

31. Competitive Inhibition

32. Kinetics of competitive inhibitor Increase [S] to overcome inhibition Vmax attainable, Km is increased Ki = dissociation constant for inhibitor

33. Vmax unaltered, Km increased

34. Non-competitive Inhibitors • Inhibitor binding site is distinct from substrate binding site. Can bind to free enzyme E and to ES • E + I = EI, ES + I = ESI or EI + S = ESI • Both EI and ESI are enzymatically inactive • The effective functional [E] (and [S]) is reduced • Reaction of unaffected ES proceeds normally • Inhibition cannot be reversed by increasing [S] • KM is not changed, Vmax is decreased by (1 + I/Ki)

35. Mixed (Noncompetitive) Inhibition

36. Kinetics of non-competitive inhibitor Increasing [S] cannot overcome inhibition Less E available, Vmax is lower, Km remains the same for available E

37. Km unaltered, Vmax decreased

38. Uncompetitive Inhibitors • The inhibitor cannot bind to the enzyme directly, but can only bind to the enzyme-substrate complex. • ES + I = ESI • Both Vmax and KM are decreased by (1+I/Ki).

39. Uncompetitive Inhibition

40. Km’ E + S ES E + P k2 + S ES2 KS1 Substrate Inhibition • Caused by high substrate concentrations

41. Substrate Inhibition • At low substrate concentrations [S]2/Ks1<<1 and inhibition is not observed • Plot of 1/v vs. 1/[S] gives a line • Slope = K’m/Vm • Intercept = 1/Vm

42. Substrate Inhibition • At high substrate concentrations, K’m/[S]<<1, and inhibition is dominant • Plot of 1/v vs. [S] gives a straight line • Slope = 1/KS1· Vm • Intercept = 1/Vm

43. 1/V I>0 I=0 1/Vm -1/Km -1/Km,app 1/[S] 1/V 1/V 1/V I>0 I>0 I=0 I=0 1/Vm,app 1/Vm,app 1/Vm 1/Vm 1/Vm -1/Km 1/[S] -1/Km 1/[S] -1/Km,app -1/Km 1/[S] Competitive Uncompetitive Substrate Inhibition Non-Competitive

44. E + S→ES→E + P + I ↓ EI E + S→ES→E + P + + II ↓ ↓ EI+S→EIS E + S→ES→E + P + I ↓ EIS ← ← ← ↑ ↑ ↑ ↑ Enzyme Inhibition (Mechanism) Uncompetitive Non-competitive Competitive E Substrate E X Cartoon Guide Compete for active site Inhibitor Different site Equation and Description [I] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [I]. [I] binds to [ES] complex only, increasing [S] favors the inhibition by [I]. [I] binds to free [E] or [ES] complex; Increasing [S] can not overcome [I] inhibition.

45. Uncompetitive Competitive Non-competitive Vmax Vmax vo Vmax’ Vmax’ I Direct Plots Km [S], mM Km’ Km [S], mM 1/vo 1/vo 1/vo I I Double Reciprocal Two parallel lines Intersect at X axis Intersect at Y axis 1/Vmax 1/Vmax 1/Vmax 1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S] Enzyme Inhibition (Plots) Vmax vo I I Km Km’ [S], mM =Km’ Vmax unchanged Km increased Vmax decreased Km unchanged Both Vmax & Km decreased I

46. Factors Affecting Enzyme Kinetics

47. Effects of pH - on enzymes - enzymes have ionic groups on their active sites. - Variation of pH changes the ionic form of the active sites. - pH changes the three-Dimensional structure of enzymes. - on substrate - some substrates contain ionic groups - pH affects the ionic form of substrate affects the affinity of the substrate to the enzyme.

48. Effects of Temperature • Reaction rate increases with temperature up to a limit • Above a certain temperature, activity decreases with temperature due to denaturation • Denaturation is much faster than activation • Rate varies according to the Arrhenius equation Where Ea is the activation energy (kcal/mol) [E] is active enzyme concentration

49. Factors Affecting Enzyme Kinetics • Temperature - on the rate of enzyme catalyzed reaction k2=A*exp(-Ea/R*T) T k2 - enzyme denaturation T Denaturation rate: kd=Ad*exp(-Ea/R*T) kd: enzyme denaturation rate constant; Ea: deactivation energy

50. REFERENCES • Michael L. Shuler and Fikret Kargı, Bioprocess Engineering: Basic Concepts (2 nd Edition),PrenticeHall, New York, 2002. • 1. James E. Bailey and David F. Ollis, Biochemical Engineering Fundementals (2 nd Edition), McGraw-Hill, New York, 1986. • www.biochem.umass.edu/courses/420/lectures/Ch08B.ppt -