Bài giảng Biochemistry 2/e - Chapter 14: Enzyme Kinetics

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

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Chapter 14Enzyme Kineticsto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll 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 Outline14.1 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 AbzymesEnzymesEnzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality Enzymes are the agents of metabolic functionCatalytic PowerEnzymes 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 !SpecificityEnzymes 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 yieldOther Aspects of EnzymesRegulation - to be covered in Chapter 15 Mechanisms - to be covered in Chapter 16 Coenzymes - to be covered in Chapter 1814.2 Enzyme KineticsSeveral terms to know! rate or velocity rate constant rate law order of a reaction molecularity of a reaction The Transition StateUnderstand 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 Enzymes do this by binding the transition state of the reaction better than the substrate Much more of this in Chapter 16!The Michaelis-Menten EquationYou 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 Understanding KmThe "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 bindingUnderstanding VmaxThe 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 dual nature of the Michaelis-Menten equationCombination 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!The turnover numberA 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 secThe catalytic efficiencyName for kcat/KmAn 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 Linear Plots of the Michaelis-Menten EquationBe able to derive these equations! Lineweaver-BurkHanes-Woolf Hanes-Woolf is best - why? Smaller and more consistent errors across the plotEnzyme InhibitorsReversible versus Irreversible Reversible inhibitors interact with an enzyme via noncovalent associations Irreversible inhibitors interact with an enzyme via covalent associations Classes of InhibitionTwo 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 inhibition14.6 Ribozymes and AbzymesRelatively 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
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