Chapter 5 (part 3): Enzyme kinetics (cont.)

Chapter 5 (part 3)Enzyme Kinetics (cont.)Michaelis-MentonVmaxKm KcatKcat/Km E + S ES E + Pk1k-1kcatVo = Vmax [S] Km + [S] VmaxKmkcatkcat/KmHow do you get values for Vmax, Km and kcat?Can determine Km and Vmax experimentallyKm can be determined without an absolutely pure enzymeKcat values can be determined if Vmax is known and the absolute concentration of enzyme is known (Vmax = kcat[Etotal]Lineweaver-Burke Plots(double reciprocal plots)Plot 1/[S] vs 1/Vo L-B equation for straight lineX-intercept = -1/KmY-intercept = 1/VmaxEasier to extrapolate values w/ straight line vs hyperbolic curveV maxKmKm ~ 1.3 mMVmax ~ 0.25 -1/Km = -0.8Km = 1.23 mM1/Vmax = 4.0Vmax = 0.25 Enzyme InhibitionInhibitor – substance that binds to an enzyme and interferes with its activityCan prevent formation of ES complex or prevent ES breakdown to E + P.Irreversible and Reversible InhibitorsIrreversible inhibitor binds to enzyme through covalent bonds (binds irreversibly)Reversible Inhibitors bind through non-covalent interactions (disassociates from enzyme)Why important?Reversible InhibitorsE + S ES -> E + PE + I EIKi = [E][I]/[EI]CompetitiveUncompetitiveNon-competitiveTypes of Reversible Enzyme InhibitorsCompetitive Inhibitor (CI)CI binds free enzyme Competes with substrate for enzyme binding.Raises Km without effecting VmaxCan relieve inhibition with more SCompetitive Inhibitors look like substratePABASulfanilamidePABA precursor to folic acid in bacteriaO2C-CH2-CH2-CO2 -------> O2C-CH=CH-CO2succinate fumarateSuccinate dehydrogenaseO2C-CH2-CO2MalonateUncompetitive Inhibitor (UI)UI binds ES complex Prevents ES from proceeding to E + P or back to E + S.Lowers Km & Vmax, but ratio of Km/Vmax remains the sameOccurs with multisubstrate enzymesNon-competitive Inhibitor (NI)NI can bind free E or ES complex Lowers Vmax, but Km remains the sameNI’s don’t bind to S binding site therefore don’t effect KmAlters conformation of enzyme to effect catalysis but not substrate binding Irreversible InhibitorsDiisopropyl fluorophosphate(nerve gas)parathionmalathionOrganophosphatesInhibit serine hydrolasesAcetylcholinesterase inhibitorsViagraKinetics of Multisubstrate ReactionsE + A + B E + P + QSequential ReactionsorderedrandomPing-pong ReactionsCleland NotationSequential ReactionsE EA (EAB) (EPQ) EQ EABPQABPQABEEAEB(EAB)(EPQ)PQEQEPEOrderedRandomPing-Pong ReactionsE (EA)(FP) (F) (FB)(EQ) EABPQIn Ping-Pong rxns first product released before second substrate bindsWhen E binds A, E changes to FWhen F binds B, F changes back to E Lineweaver-Burke Plot of Multisubstrate ReactionsIncreasing [B]Increasing [B]SequentialPing-PongVmax doesn’t changeKm changesBoth Vmax & Km change1/Vo1/[S]1/Vo1/[S]