Chapter 14. Enzyme Kinetics. to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham. Biochemistry 2/e - Garrett & Grisham

Transcription

1 Chapter 14 Enzyme Kinetics to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida

2 Outline 14.1 Catalytic Power, Specificity, Regulation 14.2 Introduction to Enzyme Kinetics 14.3 Kinetics of Enzyme-Catalyzed Reactions 14.4 Enzyme Inhibition 14.5 Kinetics of Two-Substrate Reactions 14.6 Ribozymes and Abzymes

3 Enzymes Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality Enzymes are the agents of metabolic function

4

5

6

7

8 Catalytic Power Enzymes can accelerate reactions as much as over uncatalyzed rates! Urease is a good example: Catalyzed rate: 3x10 4 /sec Uncatalyzed rate: 3x10-10 /sec Ratio is 1x10 14!

9 Specificity Enzymes selectively recognize proper substrates over other molecules Enzymes produce products in very high yields - often much greater than 95% Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield

10 Other Aspects of Enzymes Regulation - to be covered in Chapter 15 Mechanisms - to be covered in Chapter 16 Coenzymes - to be covered in Chapter 18

11 14.2 Enzyme Kinetics Several terms to know! rate or velocity rate constant rate law order of a reaction molecularity of a reaction

12

13 The Transition State Understand the difference between G and G The overall free energy change for a reaction is related to the equilibrium constant The free energy of activation for a reaction is related to the rate constant It is extremely important to appreciate this distinction!

14

15 What Enzymes Do... Enzymes accelerate reactions by lowering the free energy of activation Enzymes do this by binding the transition state of the reaction better than the substrate Much more of this in Chapter 16!

16

17 The Michaelis-Menten Equation You should be able to derive this! Louis Michaelis and Maude Menten's theory It assumes the formation of an enzymesubstrate complex It assumes that the ES complex is in rapid equilibrium with free enzyme Breakdown of ES to form products is assumed to be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S

18

19 Understanding K m The "kinetic activator constant" K m is a constant K m is a constant derived from rate constants K m is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S Small K m means tight binding; high K m means weak binding

20 Understanding V max The theoretical maximal velocity V max is a constant V max is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach V max would require that ALL enzyme molecules are tightly bound with substrate V max is asymptotically approached as substrate is increased

21 The dual nature of the Michaelis-Menten equation Combination of 0-order and 1st-order kinetics When S is low, the equation for rate is 1st order in S When S is high, the equation for rate is 0- order in S The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!

22 The turnover number A measure of catalytic activity k cat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. If the M-M model fits, k 2 = k cat = V max /E t Values of k cat range from less than 1/sec to many millions per sec

23

24 The catalytic efficiency Name for k cat /K m An estimate of "how perfect" the enzyme is k cat /K m is an apparent second-order rate constant It measures how the enzyme performs when S is low The upper limit for k cat /K m is the diffusion limit - the rate at which E and S diffuse together

25

26 Linear Plots of the Michaelis- Menten Equation Be able to derive these equations! Lineweaver-Burk Hanes-Woolf Hanes-Woolf is best - why? Smaller and more consistent errors across the plot

27

28

29

30

31 Enzyme Inhibitors Reversible versus Irreversible Reversible inhibitors interact with an enzyme via noncovalent associations Irreversible inhibitors interact with an enzyme via covalent associations

32

33 Classes of Inhibition Two real, one hypothetical Competitive inhibition - inhibitor (I) binds only to E, not to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition

34

35

36

37

38

39

40

41

42 14.6 Ribozymes and Abzymes Relatively new discoveries Ribozymes - segments of RNA that display enzyme activity in the absence of protein Examples: RNase P and peptidyl transferase Abzymes - antibodies raised to bind the transition state of a reaction of interest For a great recent review, see Science, Vol. 269, pages (1995) We'll say more about transition states in Ch 16

43

44

45

46