Chapt. 9 Regulation of EnzymesRegulation of metabolic pathwaysRegulation of glucose metabolism pathwayII. Regulation by substrate, product concentrationII. Regulation by substrate, product concentration:Different isozymes have different Km for glucoseReversible inhibitors decrease reaction velocityIII. Regulation through conformational changesA. Allosteric Activators and inhibitorsAllosteric activators and inhibitorsB. Conformational change by covalent modificationMuscle glycogen phosphorylase regulationEx. Protein kinase AOther covalent modifications affect proteinsConformational changes from Protein-Protein interactionsSmall monomeric G proteinsProteolytic cleavage is irreversibleRegulation of pathwaysLineweaver-Burk plotLineaver-Burk plots permit comparisonsKey conceptsReview questionsChapt. 9 Regulation of EnzymesRegulation of EnzymesStudent Learning Outcomes:•Explain that enzyme activities must be regulated for proper body function•Explain three general mechanisms:•Reversible binding in active site: •substrate, inhibitors •Changing conformation of active site of enzyme:•Allosteric effectors, covalent modification,•Protein-protein interactions, zymogen cleavage•(Changing concentration of enzyme)•Synthesis, degradationRegulation of metabolic pathwaysFig. 9.1Metabolic pathway analogous to cars on highway:•Flux of substrates affected by rate-limiting enzyme (barrier)•Removal of barrier increases flow•Activating rate-limiting enzymeRegulation of glucose metabolism pathwayEx. Regulation of glucose metabolism pathway:•Hexokinase & glucokinases convert glucose -> G-6-P in cells•Glycolysis for energy •Feedback regulation by ATP• Store G-6-P as glycogen•Feedforward by insulinII. Regulation by substrate, product concentrationMichaelis-Menten equation describes kinetics:More substrate gives more reaction, to maximalVi (initial velocity) relates to concentration of substrate [S] to Vmax (maximal velocity) and Km ([S] for 1/2 Vmax Applies to simple reactions:E + S  ES  E + P; k1 = forward, k2 back; k3 for E+P Vi = Vmax[S]/ Km + [S] Km = k2 + k3/k1; Vmax = k3[Et]II. Regulation by substrate, product concentration:Fig. 9.2Ex. Graph of Michaelis-Menten equation has limit of Vmax at infinite substrate.Km = [S] where Vmax/2Ex. Glucokinase Km 5 mM:If blood glucose 4 mM ->Vi = 0.44 Vmax (Vm x 4mM/ (5mM + 4 mM)Blood glucose 20 mM -> Vi = 0.8 Vmax (Vm x 20mM/ 5 + 20 mMDifferent isozymes have different Km for glucoseFig. 9.3Different hexokinases differ in Km for glucose:glucose + ATP -> G-6-P + ADPHexokinase I (rbc) only glycolysisGlucokinase (liver, pancreas) storageFasting blood sugar about 5 mM (90 mg/dL) sorbc can function even if low blood sugar of glucose S0.5 = half-max for S-shape curveReversible inhibitors decrease reaction velocityRegulation through active site: reversible inhibitorsA.Competitive inhibitors compete with substrateOvercome by excess substrate (increase apparent Km)B.Noncompetitive do not compete with substrateNot overcome by substrate (lowers [E] and Vmax)Fig. 9.4Products can also inhibit enzyme activityIII. Regulation through conformational changesRegulation through conformational changes of enzyme can affect catalytic site:•Allostery •– ex. Glycogen phosphorylase•Phosphorylation •– ex. Glycogen phosphorylase kinase•Protein-protein interactions•- ex. Protein kinase A•Proteolytic cleavage•- ex. chymotrypsinogenA. Allosteric Activators and inhibitors Allosteric enzymes:•Often multimeric, • Exhibit positive cooperativity in substrate binding (ex. Hemoglobin and O2)•T (taut state) inactive without substrate•R (relaxed) state active with substrateFig. 9.5Allosteric activators and inhibitorsFig. 9.6Allosteric enzymes often cooperative S bindingAllosteric activators and inhibitors:•Bind at allosteric site, not catalytic site•Conformational change•Activators often bind R (relaxed) state decrease S0.5•Inhibitors often bind T (taut state) increase S0.5B. Conformational change by covalent modificationFig. 9.7Phosphorylation can activate or inhibit enzymes:Protein kinases add phosphateProtein phosphatases remove•PO42- adds bulky group, negative charge, interacts with other amino acidsMuscle glycogen phosphorylase regulationFig. 9.8Muscle glycogen phosphorylase is regulated by both phosphorylation and/or allostery:•Rate-limiting step glycogen -> glucose-1-PO4• ATP use increases AMP - allostery• phosphorylation increases activity•Signal from PKAEx. Protein kinase AProtein kinase A: Regulatory, catalytic subunits:•Ser/thr protein kinase, phosphorylates many enzymes•Including glycogen phosphorylase kinase• Adrenline increase cAMP, dissociates R subunits,•Starts PO4 cascadeFig. 9.9 cAMP activates PKAOther covalent modifications affect proteinsCovalent modifications affect protein activity, location in cell:• acetyl- (on histones)•ADP-ribosylation (as by cholera toxin on G subunit)•Lipid addition(as on Ras protein)Fig. 6.13 modified amino acidsConformational changes from Protein-Protein interactionsFig. 9.10CaM kinase family activated by Ca2+/calmodulin; phosphorylate metabolic enzymes, ion channels, transcription factors, regulate synthesis, release of neurotransmitters.Ca-Calmodulin family of modulator proteins•activated when [Ca2+ ] increases. •Ca2+/calmodulin binds to targets e.g. protein kinases, allosteric resultSmall monomeric G proteins Small (monomeric) G proteinsaffect conformation of other proteins:• GTP bound form binds and activates or inhibits• GDP bound form inactive•Other intermediates regulate the G proteins (GEF, GAP, etc)•Ras family (Ras, Rho, Rab, Ran, Arf)•diverse roles in cellsFig. 9.11Proteolytic cleavage is irreversibleProteolytic cleavage is irreversible conformational change:•Some during synthesis and processing•Others after secretion: •Proenzymes inactive:•Ex. Precursor protease is zymogen: •(chymotrypsinogen is cleaved by trypsin in intestine)•Ex. Blood clotting factors fibrinogen, prothrombinRegulation of pathwaysRegulation of metabolic pathways is complex:Sequential steps, different enzymes, rate-limiting oneMatch regulation to function of path Fig. 9.12Lineweaver-Burk plotFig. 9.13Lineweaver-Burk transformation converts Michaelis-Menten to straight line (y = mx + b)•double reciprocal plot•Ease of determining Km and VmaxLineaver-Burk plots permit comparisonsLineweaver-Burk plots permit analysis