Rutgers University MBB 408 - Chapter 14 Enzyme Kinetics to accompany Biochemistry

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Chapter 14OutlineEnzymesSlide 4Slide 5Slide 6Slide 7Catalytic PowerSpecificityOther Aspects of Enzymes14.2 Enzyme KineticsSlide 12The Transition StateSlide 14What Enzymes Do....Slide 16The Michaelis-Menten EquationSlide 18Understanding KmUnderstanding VmaxThe dual nature of the Michaelis-Menten equationThe turnover numberSlide 23The catalytic efficiencySlide 25Linear Plots of the Michaelis-Menten EquationSlide 27Slide 28Slide 29Slide 30Enzyme InhibitorsSlide 32Classes of InhibitionSlide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 4114.6 Ribozymes and AbzymesSlide 43Slide 44Slide 45Slide 46Biochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyChapter 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 32887-6777Biochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyOutline•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 AbzymesBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyEnzymes•Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality •Enzymes are the agents of metabolic functionBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyCatalytic 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 !Biochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanySpecificity•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 yieldBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyOther Aspects of Enzymes•Regulation - to be covered in Chapter 15 •Mechanisms - to be covered in Chapter 16 •Coenzymes - to be covered in Chapter 18Biochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & Company14.2 Enzyme KineticsSeveral terms to know! •rate or velocity •rate constant •rate law •order of a reaction •molecularity of a reactionBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyThe 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!Biochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyWhat 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!Biochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyThe 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 SBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyUnderstanding 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 bindingBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyUnderstanding 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 increasedBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyThe 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!Biochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyThe 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 secBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyBiochemistry 2/e - Garrett & GrishamCopyright © 1999 by Harcourt Brace & CompanyThe 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 togetherBiochemistry 2/e -


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Rutgers University MBB 408 - Chapter 14 Enzyme Kinetics to accompany Biochemistry

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