Section 2.2Enzyme Enzyme KiKineticsneticsKey terms Kinetics (enzyme) Initial velocity (V0) Maximum velocity (Vmax) Substrate concentration (affinity) Steady state Michaelis constant (Km) Michaelis-Menten equation Hyperbolic plot Lineweaver-Burk plot (double reciprocal plot)1.Enzyme Kinetics As an Approach to Understanding Mechanism:(Defination and Michaelis-Menten equation)To study the rate of reaction and how it changes in response to changes in experimental parameters is called enzyme kinetics. The rate of a reaction is influenced by several factors: - concentrations of enzymes, substrates and products - modifiers like inhibitors, activators or cofactors - environmental conditions (like temperature and pH)Increase Substrate Concentration 2134567800 2 4 6 8 Substrate (mmole)Product806040200S + E P(in a fixed period of time)Juang RH (2004) BCbasicsS Affects the Rate of Enzyme-catalyzed ReactionsE + S ES E + P1.1. Initial VelocityInitial Velocity (V (V0 0) ):S is generally much grater than E2. Maximum Velocity (Vmax):the point which increases in V0 are vanishingly small as S increases.3. Pre-steady state: initial period, large excess S.4. Steady state:ES remain constantEssential of Enzyme KineticsES+P+Steady State TheorySteady State TheoryIn steady state, the production and consumption of the transition state proceed at the same rate. So the concentration of transition state keeps a constant.SEEJuang RH (2004) BCbasicsConstant ES Concentration at Steady StateSPEESReaction TimeConcentrationJuang RH (2004) BCbasicsSteady state kineticsPre-steady state kinetics Michaelis and Menten equation ( 1913; Briggs and Haldane, 1925)Steady state: Rate of ES formation = rate of ES breakdownKm is called the Michaelis constantThe maximum velocity is achieved when all the enzyme is saturated by substrate, i.e., when ES =E ThusThis is the Michaelis-Menten Equation练习P381:1,2The Michaelis-Menten Equation describes the observed velocity curveWhen S KmKm is equivalent to the substrate concentration at which V0 is Vmax/2Double Reciprocal Plot(Lineweaver-Burk Equation) y = mx + bAn Example for Enzyme Kinetics (Invertase)VmaxKmSvo1/S1 voDouble reciprocalDirect plot1)1) Use predefined amount of Enzyme E2)2) Add substrate in various concentrations S (x-axis)3)3) Measure Product in fixed Time (P/t) vo (y-axis)4)4) (x, y) plot get hyperbolic curve, estimate Vmax5)5) When y = 1/2 Vmax calculate x (S) Km1 Vmax- 1 Km1/2Juang RH (2004) BCbasicsA Real Example for Enzyme KineticsDatano 1 2 3 40.25 0.50 1.0 2.00.42 0.72 0.80 0.92Absorbance v (mmole/min)S 0.21 0.36 0.40 0.46(1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole (2) Reaction time was 10 minVelocitySubstrate ProductDouble reciprocal 1/S1/v 4 2 1 0.52.08 1.56 1.35 1.16 1.00.50vDirect plotDouble reciprocal2.01.001/v-4 -2 0 2 4 1/S0 1 2S1.0-3.8Juang RH (2004) BCbasicsEnzyme Kineticsvo=Vmax S Km + SKm VmaxE equals S； None of the above.4，Which of the following assumptions are used in the Briggs-Haldane derivation of the M-M equation? S is greater than E； The overall rate of the reaction is dependent only on the catalytic rate constant (kcat)；At saturating S, nearly all of E is found in the ES complex； ES rapidly reaches a steady state； All of the above were used in deriving the M-M equation.5，Which of the following statements about Km is correct? The Km defines the set point for S for a biochemical reaction in a cell. The Km is a measure of the affinity of S for E, only if kcat is much smaller than either k1 or k-1.The Km is S at Vmax. All of the above are true. None of the above are true.S is saturating；Many Enzyme Catalyze Reactions with Two or More SubstrateS1 + S2 P1 + P2Ping PongEKey terms Reversible inhibitor Irreversible Competitive Noncompetitive UncompetitiveSuch inhibitors are important pharmaceutical agents and useful in understanding the action mechanism of enzymesInhibitionIrreversible Reversible Competitive Noncompetitive Uncompetitive2. Inhibition kineticsA. Reversible InhibitionCompetitive Inhibitionexcerise1In competitive inhibition, inhibitor will have the following effect on the kinetics of the enzyme: A. Km will decrease.B. Vmax will stay the same.C. The reaction will cease because the inhibitor binds irreversibly.D. Km / Vmax will stay the same.Noncompetitive Inhibition(bind only to ES )Uncompetitive Inhibitionexcerise2Which type of reversible E inhibitor binds to both the free E and the ES complex? A. noncompetitive B. competitive C. uncompetitive D. Two of the above. E. None of the above. Enzyme Inhibition (Mechanism)CompetitiveNon-competitiveUncompetitiveEEDifferent siteCompete for active siteInhibitorSubstrateCartoon GuideEquation and DescriptionI I binds to free E