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Chapter 14

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Chapter 14

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  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 32887-6777

  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. Catalytic Power • Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates! • Urease is a good example: • Catalyzed rate: 3x104/sec • Uncatalyzed rate: 3x10 -10/sec • Ratio is 1x1014!

  5. 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

  6. 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

  7. 14.2 Enzyme Kinetics Several terms to know! • rate or velocity • rate constant • rate law • order of a reaction • molecularity of a reaction

  8. 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!

  9. 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!

  10. The Michaelis-Menten Equation You should be able to derive this! • Louis Michaelis and Maude Menten's theory • It assumes the formation of an enzyme-substrate 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

  11. Understanding Km The "kinetic activator constant" • Km is a 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

  12. Understanding Vmax The theoretical maximal velocity • Vmax is a constant • Vmax is the 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

  13. 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!

  14. 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, k2 = kcat = Vmax/Et • Values of kcat range from less than 1/sec to many millions per sec

  15. 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

  16. 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

  17. Enzyme Inhibitors Reversible versus Irreversible • Reversible inhibitors interact with an enzyme via noncovalent associations • Irreversible inhibitors interact with an enzyme via covalent associations

  18. 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

  19. 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