1. Quiz #3 Define Enzyme Classes Systematic naming –Given a reaction (including names) –Use subclass designation if appropriate Catalytic mechanisms –Define –How does a given amino acid participate? Discuss protonation state and reaction.

2. Enzyme Kinetics The study of the rates at which enzyme-catalyzed reactions occur.

3. Value of Enzyme Kinetics Quantitation of Enzyme Activity –Purification Competing Substrates and Inhibitors –Suggest physiological functions –Suggest regulatory mechanisms Response to Different Conditions –Reaction mechanism

4. Kinetic Data plus Structure and Mechanism Clues to an enzyme’s biological function Ways to modify an enzyme for therapeutic purposes

5. Enzyme-Catalyzed Reactions are Reversible (Accelerate Attainment of Equilibrium)

6. Elementary Reactions A ——> P Reaction Intermediates A ——> I 1 ——> I 2 ——> P

7. Two-Step Reactions (Rate Determining Step)

8. Chemical Kinetics Can reveal the number of reacting species Can reveal the rate of their interactions Are described by Rate Equations

9. Rate Equations Reaction Order or Molecularity: number of molecules participating in a reaction Velocity (V): molar per second (Ms –1 ) Rate Constant (k): proportional to frequency of interaction between reactants

10. Rate Equations (General Form) aA + bB ——> P V = k[A] a [B] b k = rate constant Order of Reaction = a + b

11. Reaction Order (Reaction Molecularity) First-order or unimolecular Second-order or bimolecular Third-order very rare 4 th -order are unknown

12. Rates of Reactions First-Order Reactions

13. Time Course of Reaction

14. Kinetics of Reactions

15. First-Order Reaction

16. Initial Velocity (V O ) V = k F [S] – k B [P] Initial Velocity (V o ) ([P] = 0) V o = k F [S]

17. Uncatalyzed Reaction

18. Enzyme Catalysis Hyperbolic Relationship V O not proportional to [S]

19. Michaelis-Menten Formulation

20. Enzyme Kinetics Experiment: (1)Mix enzyme + substrate (2)Record rate of product formation as a function of time (the velocity of reaction) (3) Plot initial velocity versus substrate concentration. (4) Change substrate concentration and repeat

21. Postulated Pathway of Enzyme Catalysis: Formation of Enzyme-Substrate Complex

22. Assumptions: Initial Velocities Catalysis is rate limiting V o = k 2 [ES] (rate limiting)

23. Assumptions Binding equilibrium (k –1 >>k 2 ) Steady state (d[ES]dt = 0)

24. Assumptions Binding equilibrium (k –1 >>k 2 ) Steady state (d[ES]dt = 0)

25. Additionally [s]>>>>>>>>>[E-S] –[S] doesn’t change during initial velocity [E T ] = [E F ] + [E-S]

26. Progress Curves (M-M Enzyme) Steady-State Very short

27. Michaelis-Menten Equation V O = V max [S] K m + [S] Assumes All Enzyme Molecules are Catalytically Active

28. Michaelis Constant K M is the substrate concentration at which the reaction velocity is half-maximal High affinity = low K m ; Low Affinity = high K m K m = k -1 + k 2 k 1

29. Michaelis-Menton Enzyme Kinetics

30. Catalytic Constant (Turnover Number) # of substrate converted to product # of enzyme = Unit of time V max = k cat X # of enzymes # can be any units (molecules, moles, μmoles) But all must be the same units

31. Catalytic Efficiency k cat /K M –Limited by diffusion (10 8 to 10 9 M -1 s -1 )

32. Lineweaver-Burk Formulation

33. Lineweaver-Burk Plot

34. Eadie-Hofstee Formulation

35. Eadie-Hofstee Plot

36. A certain enzyme catalyzes the conversion of 100 μmoles of A to B in one second, when the concentration of A is 20 mM. It catalyzes the conversion of 350 μmoles of A to B in one second when the concentration of A is either 500 mM or 550 mM. -Calculate K m -Draw the Michaelis-menton curve and Lineweaver-Burk plot for this enzyme -Can you calculate turnover #?