Biochemistry 2e Garrett Grisham Chapter 14 Enzyme Kinetics

Biochemistry 2/e - Garrett & Grisham 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 32887 -6777 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Enzymes • Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality • Enzymes are the agents of metabolic function Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Catalytic Power • Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates! • Urease is a good example: – Catalyzed rate: 3 x 104/sec – Uncatalyzed rate: 3 x 10 -10/sec – Ratio is 1 x 1014 ! Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 14. 2 Enzyme Kinetics • • • Several terms to know! rate or velocity rate constant rate law order of a reaction molecularity of a reaction Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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! Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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! Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Understanding Km • • The "kinetic activator constant" Km is a constant derived from rate constants Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S Small Km means tight binding; high Km means weak binding Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Understanding Vmax • • The theoretical maximal velocity Vmax is a constant Vmax is theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate Vmax is asymptotically approached as substrate is increased Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham The dual nature of the Michaelis-Menten equation Combination of 0 -order and 1 st-order kinetics • When S is low, the equation for rate is 1 st 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! Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham The turnover number A measure of catalytic activity • kcat, 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 = kcat = Vmax/Et • Values of kcat range from less than 1/sec to many millions per sec Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham The catalytic efficiency • • Name for kcat/Km An estimate of "how perfect" the enzyme is kcat/Km is an apparent second-order rate constant It measures how the enzyme performs when S is low The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Linear Plots of the Michaelis. Menten Equation • • Be able to derive these equations! Lineweaver-Burk Hanes-Woolf is best - why? Smaller and more consistent errors across the plot Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Enzyme Inhibitors Reversible versus Irreversible • Reversible inhibitors interact with an enzyme via noncovalent associations • Irreversible inhibitors interact with an enzyme via covalent associations Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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 1835 -1842 (1995) – We'll say more about transition states in Ch 16 Copyright © 1999 by Harcourt Brace & Company