02/15/2007 05:12 PMLEC15_EnzReg1Page 1 of 10http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC15_EnzReg1/LEC15_EnzReg1.htmlLecture 15: Enzymes: Regulation 1 [PDF]Reading: Berg, Tymoczko & Stryer, Chapter 10, pp. 275-282Updated on: 2/15/07 at 5:00 pm Key ConceptsAmounts of many key enzymes are regulated at the level of control of transcription, mRNA processing, and/or translation(mechanisms covered in BIOC 411 or BIOC 461), or destruction (proteolytic degradation) of old/unwanted enzymes. Activities of many key enzymes are regulated in cells, based on metabolic needs/conditions in vivo.Regulation of enzyme activity can increase or decrease substrate binding affinity and/or kcat.5 principal ways protein activity (including enzyme activity) is regulated:allosteric controlinteraction with regulatory proteinsmultiple forms of enzymes (isozymes)reversible covalent modificationirreversible covalent modification, including proteolytic activation Allosteric Controlconformational changes -- 2 conformations, "R" (more active) and "T" (less active)allosteric activators (positive effectors/modulators)allosteric inhibitors (negative effectors/modulators)Allosterically regulated enzymes always multi-subunitATCase as an examplehomotropic effector (substrate aspartate)heterotropic effectors (activator = ATP; inhibitor = CTP)2 models for allosteric proteinsMWC model (Monod-Wyman-Changeux, the "concerted" model):Either all subunits in a given protein molecule are in R state or all are in T state; no hybrid enzyme molecules.ATCase fits this modelKoshland model (KNF model, the "sequential" model):includes possibility of "hybrid" enzyme molecules with some subunits in R state and others in T state.Hemoglobin (next lecture) requires elements of this more complicated modelObjectivesList 5 general strategies used by cells for biological regulation of enzyme activity.Explain the meaning of the “first committed step” in a metabolic pathway, and of the “rate-limiting step” in a metabolic pathway(which frequently turn out to be the same step), and explain the significance of those terms in regulation of “flow” of moleculesthrough that pathway.Define/explain the following terms (some are review): quaternary structure, multimeric protein, homopolymeric protein,heteropolymeric protein, ligand, binding site, fractional saturation, feedback inhibition, cooperativity, cooperative binding, allosteric(allosteric site, allosteric effector/regulator, allosteric protein...), effector/regulator, homotropic effector/regulator, heterotropiceffector/regulator, allosteric activator (positive heterotropic effector/regulator), allosteric inhibitor (negative heterotropiceffector/regulator), protomer, prosthetic group.Briefly explain the allosteric regulation ofATCase, including its quaternary structure, its role in metabolism, and how its activityis regulated by allosteric inhibition and activation. Include the physiological rationale for the inhibition and activation.Sketch plots of Vo vs. [S] for an allosteric enzyme that illustrate positive homotropic regulation and positive and negativeheterotropic regulation, with ATCase as an example. Specifically, sketch (all on the same axes) for ATCase: Vo vs. [aspartate]curves with no heterotropic regulators present, with an allosteric inhibitor present, and with an allosteric activator present.02/15/2007 05:12 PMLEC15_EnzReg1Page 2 of 10http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC15_EnzReg1/LEC15_EnzReg1.htmlBriefly explain the difference between the concerted model and the sequential model for an allosteric enzyme, in terms of R and Tconformations of the individual subunits and of the whole quaternary structure, and whether “hybrid” quaternary structures(with some subunits in the T conformation and some subunits in the R conformation) exist in each model.5 principal ways protein activity (including enzyme activity) is regulated:1. Allosteric controlRegulation of binding affinity for ligands, and/or of catalytic activity, by conformational changes caused by binding of thesame or other ligands at other sites on protein ("allosteric effects")Changes involve simple association/dissociation of small molecules, so enzyme can cycle RAPIDLY between active andinactive (or more and less active) states.2. Interaction with regulatory proteinsBinding of a different protein to the enzyme alters the enzyme activity (activates or inhibits the enzyme) 3. Multiple forms of enzymesIsozymes (isoenzymes) = multiple forms of enzyme that catalyze same reaction but are products of different genes (sodifferent amino acid sequences)Isozymes differ slightly in structure, and kinetic & regulatory properties are differentCan be expressed in different tissues or organelles, at different stages of development, etc.4. Reversible covalent modificationModification of catalytic or other properties of proteins by enzyme-catalyzed covalent attachment of a modifying group.Modifications removed by catalytic activity of a different enzyme, so enzyme can cycle between active and inactive (or moreand less active) states.5. Proteolytic activationIrreversible cleavage of peptide bonds to convert inactive protein/enzyme to active form.Inactive precursor protein = a zymogen (a proenzyme).Proteolytic activation IRREVERSIBLE, but eventually the activated protein is itself proteolyzed, or sometimes a tight-bindingspecific inhibitory protein inactivates it.1. ALLOSTERIC REGULATION(Introductory material here is a review of concepts in chapter 7, ligand binding and allosteric regulation of hemoglobin, and won't becovered much in class.) Allosterically regulated enzymes: don't follow Michaelis-Menten kinetics (Vo vs. [S] is not a hyperbola.)multisubunit enzymes (more than one catalytic subunit, so > 1 active site/enzyme molecule)cooperative substrate binding: binding of substrate to 1 active site affects properties of other binding sites (on othersubunits) of the same enzyme moleculeResult: a sigmoid Vo vs. [S] curve (NOT hyperbolic) -- diagnostic of COOPERATIVITYVelocity increases steeply in [S] range around the apparent "KM" ([S] where velocity = 1/2 Vmax), so in that substrateconcentration range (around KM), a small change in [S] makes a big change in velocity.Regulatory consequences: binding another molecule (an allosteric inhibitor or activator) canSHIFT whole curve to RIGHT:Allosteric inhibitor --> velocity is less at a given [S], requires more [S] to reach
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