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

Biochemistry 2/e - Garrett & Grisham

Copyright © 1999 by Harcourt Brace & Company

Enzyme Kinetics



Enzymes

• Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality

• Enzymes are the agents of metabolic function



Catalytic Power

• Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates!



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





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!





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









Enzyme Kinetics

Several terms to know!

• rate or velocity

• rate constant

• rate law

• order of a reaction



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





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







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



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





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







The catalytic efficiencyName 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







Enzyme Inhibitors

Reversible versus Irreversible

• Reversible inhibitors interact with an enzyme via noncovalent associations

• Irreversible inhibitors interact with an enzyme via covalent associations



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



Competitive inhibition



Non-competitive inhibition

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