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BU BIOL 302 - Regulatory Strategies
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BIOL 302 1st Edition Lecture 16 Outline of Last Lecture I. Zymogens, II. FibrinIII. Carbonic AnhydraseIV. Restriction EnzymesV. Myosin and ATP HydrolysisOutline of Current Lecture VI. Regulatory Strategies Current LectureI. Clicker question: Which one is the Hill plot of myoglobin?These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.II. O2 bindingA. Hemoglobin & MyoglobinIII. Regulatory StrategiesA. Cell growth and survival depend on spatial and temporal control of a vast array of biochemical processes.B. Enzymes are regulated in multiple ways, including1. Allosteric control2. Isozymes3. Stimulation or inhibition by regulatory proteins4. Reversible covalent modification5. Irreversible covalent activationIV. Allosteric EnzymesA. Allosteric enzymes are generally larger and more complex than non-allosteric enzymes. B. Have more than one binding site, where effector binding at one site induces a conformational change, altering affinity at another site.C. An allosteric activatorincreases enzyme activity, an allosteric inhibitor decreases its activity.1. Homotropic – when the effector is the substrate2. Heterotropic - when the effector is a molecule other than the normal substrateV. Allosteric enzymes control metabolic fluxA. An allosteric enzyme, early in a metabolic pathway, is inhibited by an end-product. Often takes place at the committed step of the pathway, the stepwhich commits a metabolite to a pathway.VI. Allosteric EnzymesA. Allosteric enzymes play a pivotal role in cells because they have two functions – they not only catalyze reactions in metabolic pathways, but also control the rates of these pathways.B. All allosteric enzymes* consist of more than one subunit (polypeptide chains)VII. The Kinetic Properties of Allosteric Enzymes Diverge from Michaelis-Menten BehaviorA. The relationship between enzyme velocity and substrate concentration is often a sigmoidal saturation curve for an allosteric enzyme rather than hyperbolic (does not obey Michaelis Menten kinetics)VIII. Kinetic properties of regulatory enzymesA. Look at dependence of initial velocity as a function of [S]B. At low [S], velocity is weakly responsive to change in [S]C. As [S] increases, a point is reached at which velocity steeply rises in response to small increase in [S]D. This is an example of cooperative bindingIX. Aspartase Transcarbamoylase, an allosteric enzymeX. Allosteric RegulationA. ATCase catalyzes first step of the pathwayB. UMP, UTP, CTP are end products of the pathway; slow down first step of pathwaywhen final products are in plentiful supplyXI. Kinetic Evidence for Allosteric RegulationA. T-> R switch induced by the substrate is called a positive homotropic effectXII. Heterotropic allosteric inhibition of ATCaseby CTPA. CTP inhibits ATCase. Bindingis noncovalent and readilyreversible; if the CTP concentration decreases,the rate of N-carbamoylaspartate increases. ATCase activity responds rapidly and reversibly to fluctuations in the cellular concentration of CTP.XIII. Heterotropic allosteric activation of ATCase by ATP A. When [ATP] is high enough, the shape of the curve approaches that of a hyperbolic curve. This means binding of ATP to enzyme stabilizes the R state.B. Later, found ATP competes with CTP for binding to the regulatory subunitsXIV. Compare ATP and CTP effectsXV. Clicker question: What is the advantage of evolving an ATCase enzyme that is activated by ATP? A. CTP is a precursor from which ATP is made.B. ATP is a precursor from which CTP is made.C. This will balance the purine and pyrimidine pools.D. ATP is used as a signaling molecule in several pathways. E. ATP concentration is increased after intracellular Calcium is increased.XVI. Compare ATP and CTP effectsA. Y – fractional activity - is the fraction of active sites bound to substrate. B.Y is direct proportional to reaction velocity.XVII. Allosteric RegulationA. What is the molecular basis of allosteric control?1. Gerhart & Schachman disrupted quaternary structure by reaction of Cys –SHwith mercurial reagents, then separated two parts by ultracentrifugation.XVIII. Allosteric RegulationA. When ATCase was disrupted, onefragment had catalytic activity(hyperbolic curve) and was notinhibited by CTP: Catalytic trimers(c3) B. The other fragment had nocatalytic activity but could bindCTP: Regulatory dimers (r2)C. When catalytic trimers areseparated from regulatory dimers,result is hyperbolic curve for vi vs[Asp]XIX. ATCase StructureA. ATCase has two catalytic trimers (yellow), each contains 3 active sitesB. ATCase also has three regulatory dimers (red)with sites for bindingCTPC. Interaction of catalytic and regulatory subunits lead to the allosteric regulation of this enzymeXX. The R state and the T state are in equilibrium. A. Even in the absence of SB. ATCase is in equilibrium between the R state and the T state.XXI. Allosteric RegulationA. CTP inhibition:B. CTP binds to the regulatory subunits stabilizing the T stateC. CTP is a heterotropic allosteric inhibitorXXII. Clicker question: The T- to R state transition in ATCase uses theA. Sequential mechanismB. Concerted mechanismC. Ping pong kineticsD. Ordered bindingE. all of the aboveXXIII. Allosteric regulationXXIV. Regulation: IsozymesA. Different enzymes that catalyze the same reaction1. have different amino acid sequences2. coded for by separate but related genesoftenarise bygeneduplication and divergence3. distinguished from related enzymes that arise by alternative splicing, called isoforms4. permits fine-tuning of metabolism to meet needs of diverse tissues or developmental stagesXXV. IsozymesA. Isozymes differ in various ways:1. Kinetic parameters – differ in Km, Vmax2. Regulatory parameters: differin sensitivity to inhibitors and activators3. Adapted to functioninga. In different tissues of multicellular organismb. Targeted to different subcellular compartmentsc. Expressed during different developmental stagesXXVI. Covalent ModificationA. Regulatory enzymes can be transformed from active to inactive forms by chemical modification1. Mostly catalyzed by other enzymes2. Most modifications are reversibleXXVII. Covalent ModificationA. Functional Consequences1. Modify reactivity of active-site residue2. Mediate folding3. Prevent or Promote Degradation4. Alter interactions with other proteins5. Impair substrate binding6. (right)7. Tether


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BU BIOL 302 - Regulatory Strategies

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