02/04/2007 09:12 PMLEC11_EnzymeKineticsPage 1 of 11http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC11_EnzKin/LEC11_EnzKin.htmlLecture 11: Enzymes: Kinetics [PDF]Reading: Berg, Tymoczko & Stryer, Chapter 8, pp. 216-225Updated on: 2/4/07 at 9:00 pm Key ConceptsKinetics is the study of reaction rates.Study of enzyme kinetics is useful for measuring concentration of an enzyme in a mixture (by its catalyticactivity), its purity (specific activity), measurement of the catalytic efficiency and/or the specificity of an enzymefor different substrates, comparison of different forms of the same enzyme in different tissues or organisms,effects of inhibitors (which can give information about catalytic mechanism, structure of active site, potentialtherapeutic agents...)Dependence of velocity on [substrate] is described for many enzymes by the Michaelis-Menten equation:kinetic parameters:Km (the Michaelis constant)kcat (the turnover number, which relates Vmax, the maximum velocity, to [Et], the total active siteconcentration)kcat/Km (the catalytic efficiency of the enzyme) -- can't be greater than limit imposed by diffusion control,~108-109 M-1sec-1Kinetic parameters can be determined graphically by measuring velocity of enzyme-catalyzed reaction atdifferent concentrations of substrate.Learning ObjectivesTerminology: active site, enzyme-substrate complex, induced fit, initial velocity, steady state, Vmax , KM, kcat , turnover number, KES , enzyme efficiency.Write out a simple Michaelis-Menten kinetic mechanism for an enzyme-catalyzed reaction.Recognize the Michaelis-Menten equation, and sketch a graph of Vo vs. [S] for an enzyme-catalyzedreaction that illustrates Vmax and KM.Explain the definition of KM in terms of the rate constants in the Michaelis-Menten kineticmechanism; give the operational definition of KM that holds no matter what the actual kineticmechanism is for a particular enzyme.Explain the relationship of kcat to Vmax, and the relationship of KM to KES.State the units of KM, kcat, and Vmax.Express the ratio of occupied active sites to total enzyme active sites ([ES]/[ET]) in terms of Vo andVmax. What is the maximum possible value of that ratio?02/04/2007 09:12 PMLEC11_EnzymeKineticsPage 2 of 11http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC11_EnzKin/LEC11_EnzKin.htmlGiven a plot of Vo/Vmax vs. [S], find the value of KM from the plot.What two things is the parameter kcat/KM used to indicate? What sets the upper limit for the valueof kcat/KM for an enzyme, and what is the approximate range of numerical values for that upperlimit of kcat/KM, with units?Enzyme KineticsREVIEW: How do enzymes reduce the activation energy (ΔG‡)?1) by lowering the free energy of the transition state (‡), e.g., by binding the transition state tightly2) by changing the reaction pathway by which reactants react to form productse.g., taking a 1-step uncatalyzed reaction and accomplishing the same result by a different route, with severalintermediate reactions en route.Each reaction step has its own transition state with its own activation energy (ΔG‡).If all of the individual steps' ΔG‡s are lower than the activation energy of the uncatalyzed reaction, theoverall rate of product formation will be greater in the presence of the catalyst.The overall rate of the catalyzed reaction is dictated by the slowest step in a multistep reaction.Given a free energy diagram like the one in Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed.(2004) Fig. 6-3 (previous lecture notes), how do you identify the rate-limiting (slowest) step on thereaction coordinate?BINDING = the essence of enzyme action!binding of SUBSTRATE to form an ES COMPLEXbinding of TRANSITION STATE more tightly than the substrateBinding occurs at ACTIVE SITE of enzyme.Subsequent chemical events can then occur.Active site:relatively small part of whole enzyme structure3-dimensional cleft with participating components from different parts of primary structurewater often excluded so substrates and intermediates are in non-aqueous environment (unless H2O is a reactant)Binding uses multiple weak interactions:hydrogen bondssalt linksvan der Waals interactionshydrophobic effect(We'll come back to binding and reduction of ΔG‡ when we discuss enzyme mechanisms, how (chemically) enzymescatalyze reactions.)Berg, Tymoczko & Stryer, 6th ed., Fig. 8.5: Structure of an ES complex (a cytochrome P450 bound tocamphor)02/04/2007 09:12 PMLEC11_EnzymeKineticsPage 3 of 11http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC11_EnzKin/LEC11_EnzKin.htmlBerg, Tymoczko & Stryer,6th ed. Fig. 8.7: Lysozyme,with color coded active siteresidues that come fromdifferent parts of AAsequenceSPECIFICITY of bindingdepends on active site crevice being sterically and chemically precisely complementary to the groups it isbinding (best complementarity may be present in E•S complex but NOT in free enzyme -- induced fit) Enzymes flexible -- conformational changes can occurwhen substrate bindsduring the reaction, to get maximal complementarity to the transition stateinduced fit: conformational changes giving tighter binding in a new conformationBerg, Tymoczko & Stryer, 6th ed. Fig. 8.9: old "Lock andkey" model for E-S interaction Berg, Tymoczko & Stryer, 6th ed. Fig. 8.10: Induced fitFor many (probably MOST) enzymes, the active site02/04/2007 09:12 PMLEC11_EnzymeKineticsPage 4 of 11http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC11_EnzKin/LEC11_EnzKin.htmlActive site of free enzyme complementary to shape of Seven without substrate bound.assumes shape complementary to S only when S is bound.Why study enzyme kinetics (reaction rates)?compare enzymes under different conditions, or from different tissues or organismsunderstand how differences relate to physiology/function of organisme.g., physiological reason for different Km values for hexokinase and glucokinase -- discussed later in coursecompare activity of same enzyme with different substrates (understand specificity)measure amount or concentration of one enzyme in a mixture by its activitymeasurement of VELOCITY = reaction ratemeasure enzyme purity (specific activity = amount of activity/amount of protein)study/distinguish different types of INHIBITORS*info about enzyme active sites & reaction mechanism*development of specific DRUGS (enzyme inhibitors)SIMPLE ENZYME-CATALYZED REACTION:Measurement of velocityV = rate of
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