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

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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Catalytic Power Enzymes can accelerate reactions as much as 10 16 over uncatalyzed rates!

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

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

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

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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company What Enzymes Do.... Enzymes accelerate reactions by lowering the free energy of activation by binding the transition state of the reaction better than the substrate

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

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

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

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

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

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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The dual nature of the Michaelis-Menten equation Combination of 0-order and 1st-order kinetics The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S ! 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

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

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

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

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

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

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

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

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

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

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

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

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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Classes of Inhibition Competitive inhibition - inhibitor (I) binds only to E, not to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES

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

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

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