1Chapter 8 (continued)EnzymesYou should read the entire chapter. For the midterm, you will not be tested on the following topics :1. kinetic details of different types of enzyme inhibition (pp. 266-268) 2. kinetic details of allosteric enzymes (pp. 280, middle of page, to 281, middle of page).3. Details of regulation by phosphorylation (pp. 282, near bottom of page, to 286, near bottom of page).4. Hexokinase and Enolase--but these will be included later, when we study glycolysisChymotrypsinVmax[S]Vo =____________KM + [S]KM = [S]when Vo = Vmax_____2Enzyme Kinetics:Michaelis-Menton EquationDouble-Reciprocalor Lineweaver-Burk Plot1 KM 1 ______ = _______ +______Vo Vmax[S] VmaxE + S ES E + Pk1k-1k2Vmax = k2[Etotal]This is true only for this specific (rather simple) mechanism!Vmax[S]Vo =____________KM + [S]2Vo =Vmax[S]_________Km + [S]=kcat[Etotal][S]_________________Km + [S]kcator Turnover NumbersVmax = kcat[Etotal]More general:Catalytic efficiency, kcat/KmMeasures how the enzyme performs when S is lowThe upper limit for kcat/Kmis the diffusion limit - the rate at which E and S diffuse togetherkcat[Etotal][S]_________________Km + [S]Vo=kcat[Etotal][S]_________________Km=when S is lowMany enzymes catalyze reactions with two or more substrates a. Mechanisms involving ternary complexb. Ping-pong or double-displacement reaction mechanisms3Ternary ComplexReaction Mechanism Ping-pongorDouble-Displacement Reaction MechanismsSteady-State Kinetics Can Help Determine MechanismInhibitor (I) binds only to E, not to ESInhibitor (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 inhibitionInhibitor (I) binds either to E and ES or to ES alone.Enzyme InhibitionCompetitive Uncompetitive MixedInhibition Inhibition InhibitionKmchanges while Vmaxdoes not Km and Vmaxboth changeKm and Vmaxboth changeDouble-Reciprocalor Lineweaver-Burk Plot1 KM 1 ______ = _______ +______Vo Vmax[S] Vmax4Irreversible Inhibitionchymotrypsindiisopropylfluorophosphate(Irreversible)Enzyme Activity is Affected by pHStructure of Chymotrypsin56Pre-steady state evidence for an acyl enzyme intermediate7Regulatory Enzymesimportant in controlling flux through metabolic pathways2. Regulation by covalent modification1.Allosteric enzymesExample of an allosteric regulatory enzymeTwo views of aspartate transcarbamoylase•12 subunits: 6 are catalytic and six are regulatory•Regulatory subunits are shownin red and yellowConversion of L-threonine to L-isoleucine catalyzed by a sequence five enzymes, E1-E5L-isoleucine is an inhibitoryallosteric modulator of E1Regulation by Feedback Inhibition82. Regulation by covalent modificationMost common2. Regulation by Covalent Modification:Proteolytic cleavage of
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