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UVM BIOC 205 - Enzymes and Kinetics

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Enzymes & Kinetics IVRegulation and AllosteryMargaret A. DaughertyFall 2003ENZYME-SUBSTRATE INTERACTIONSTHE LOCK & KEY MODELA perfect match between enzyme and substratecan explain enzyme specificity does not explain enzymatic catalysisENZYME-SUBSTRATE INTERACTIONSTHE INDUCED FIT MODELKEY FEATURES:ENZYME STRUCTURE CHANGES IN PRESENCE OF SUBSTRATE --- BRINGS CATALYTIC GROUPS INTO CORRECT POSITION TO DO CHEMISTRY ---SUBSTRATE IS FORCED INTO TRANSITION STATE CONFORMATIONGENERAL THOUGHTS ON REGULATION1). Enzymes slow down as product accumulates2). Availability of substrates (and cofactors) determines reaction rate3). Enzymes are controlled at the level of DNA4). Many enzymes are regulated via reversible covalent modification5). Many enzymes regulated via non-covalent interactions with small moleculesBIOC 205REGULATION OF ENZYME ACTIVITY?How does the cell know when “enough is enough”?hexokinaseFirst step in glycolysis:hexokinaseBIOC 205Substrate-level controlREGULATION OF ENZYME ACTIVITY?Problem: Most metabolic pathways involve manyenzymes that act sequentiallyE1ABCDE2E3 E5E4threoninedehydratasethreonineleucineFeedback inhibition: the end product of the pathwaybinds to, and inhibits a regulatory enzyme on the pathway.BIOC 205FEEDBACK CONTROL:INHIBITION & ACTIVATIONIncrease in G can inhibit formation of D, activateformation of K. Similarly N can inhibit formationof K and activate formation of D.The same substrate can act as an inhibitor of onepathway, and an activator of a second pathway.+BIOC 205REGULATORY ENZYMES: THREE GENERAL CLASSES1). Enzymes regulated via reversible covalentmodification;2). Enzymes regulated via proteolytic cleavage.3). “Other” types: isozymes, modulator proteins4). Allosteric enzymesRegulatory enzymes: enzymes that control key metabolicpoints in a pathway. Usually located at the first committedstep to a pathway.BIOC 205ENZYMES REGULATED VIA COVALENT MODIFICATIONModification AA residue30-50%BIOC 205ENZYMES REGULATED VIA COVALENT MODIFICATIONGlycogen synthase: multiple phosphorylation sites,that can act independently, or in concert.(p) of any or all ofthese sites moderatelyaffects activity(p) of all sitesdramatically affectsactivity(p) of site 5 does notaffect activityBIOC 205Secretions from exocrine pancreas play roleHCO3-: Neutralizes stomach acidityZymogens of proteases:Proteases secreted in inactive formTrypsinogenChymotrypsinogenProlelastaseProcarboxypeptidasesCleaved to active formENZYMES REGULATED VIA PROTEOLYSIS:Proteases in protein digestionBIOC 205ACTIVATION OF INTESTINAL PROTEASESNOTE: These all work on one another, so they all must be activated in ashort time span! Regulation is key in everything!BIOC 205ENZYMES REGULATED VIA PROTEOLYSIS:THE BLOOD CLOTTING CASCADEBIOC 205ISOZYMES: LACTATE DEHYDROGENASEIsomer: one or more quaternary forms; differ in ratios of catalyticsubunits that make up the quaternary structure.BIOC 205MODULATOR PROTEINS: Influenceactivity of an enzymeModulator proteins interact directly with an enzymeCan either upregulate or downregulate activitycAMP regulatory protein: dimer of C (catalytic subunit) and R(regulatory subunit). Dissociation of R allows activation of CBIOC 205• Multi-subunit proteins that can have different quaternarystructures (T - low affinity; R - high affinity)• Situated at key steps in metabolic pathways (1rst step)• Enzymes that are regulated by the binding of an effectormolecule, i.e., a signal molecule that can influence the actionof the enzyme.• Effectors bind at an allosteric site. (Site that is not theactive site)• Allosteric effectors can be;–Postive effectors - increase enzyme rate–Negative effectors - decrease enzyme rate• Enzymes can posses both positive and negative regulatorysitesALLOSTERIC ENZYMES: KEY FACTSBIOC 205Allosteric Enzymes: Substrate BindingSigmoidal curve - homo-allostery (cooperative binding)Lineweaver-Burke plot is non-linearBIOC 205 BIOC 205Symmetry Model: MWC Monod - Wyman - Changeux ModelR: conformation is high-affinity; favors bindingT: conformation is low-affinity; disfavors bindingIn R:All subunitshave RconformationIn T:All subunitshave TconformationState 1: unligated stateBIOC 205Symmetry Model: MWC Monod - Wyman - Changeux ModelState 1: unligated stateKT and KR: substrate dissociation constants equilibrium constant for 4o switchKT= [E][S]/[ES]Model assumes KT >> KR;This means R has greateraffinity for substrate - doesn’t disociate aseasilyTwo types of equilibrium constants: L and KT/KR Increasing L favors T;Harder to switch;More sigmoidalR <--> TES <--> E + S A larger c means that substratedissociates less from R than T (R hashigher affinity); more hyperbolicc = KR/KTBIOC 205Symmetry Model: MWC Monod - Wyman - Changeux ModelState 2: Ligated stateS= substrate binding siteF = effector binding siteBinding of one substrate: 4o shifts to favor R R is the high affinity structure All binding sites now high affinity Makes binding of next ligand easierPOSITIVE COOPERATIVITY!Homotropic: that like moleculesinfluence binding of like moleculesBIOC 205BIOC 205Symmetry Model: MWC Monod - Wyman - Changeux ModelHeterotropic effectors: small molecules that influencethe binding of substrate; work by binding at a site otherthan the substrate binding site;Positive effectors: moleculesthat favor the high affinity RconformationNegative effectors: moleculesthat favor the low affinity TconformationEffector binding siteBIOC 205Effect of positive & negative effectors on binding curvesBIOC 205K vs. V SystemsK system: The K0.5changes +/- allostericeffectors; Vmax is unchangedV system: Vmax changes +/-allosteric effectors K0.5 is unchangedT and R have same affinity for substrate;Differ in catalytic abilityDiffer in affinities for activator and inhibitorT and R have differentaffinities for substrate,activator and inhibitorBIOC 205An example: Glycogen phosphorylaseRole: breaks glycogen down to glucoseWhere: liver and muscleWhen: Times of energy need (mild starvation, exercise)BIOC 2051). Active site (PLP); Pi2). Glycogen storage site3). Allosteric effector site4). Regulatory site1234Dimer: 2 copies of each siteBIOC 205Control of GP1). Response to fuel needs:High fuel state:Enzyme “off”high ATPhigh Glucosehigh G6PLow fuel state:Enzyme “on”high AMPlow ATP…2). Covalent modificationStress situation!Molecule on!BIOC 205v vs. S curves for glycogen phosphorylaseBinding ofsubstrate, Pi,shows positivehomotropiccooperativityBinding


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