BIOC 460, Spring 2008LEC 15, Enzymes - Regulation 1 1Lecture 15Enzymes: Regulation 1Allosteric RegulationIsozymes•Subsequent 2 lectures:– Reversible covalent modification– Association with regulatory proteins– Irreversible covalent modification/proteolytic cleavageReading: Berg, Tymoczko & Stryer, 6th ed.,Chapter 10, pp. 275-283Key Concepts• Amounts of many key enzymes are regulated at the level of control oftranscription, mRNA processing, and/or translation (mechanisms coveredin BIOC 411 or BIOC 461), or destruction (proteolytic degradation) ofold/unwanted enzymes.• Activities of many key enzymes are regulated in cells, based onmetabolic needs/conditions in vivo.• Regulation of enzyme activity can increase or decrease substratebinding affinity and/or kcat.• 5 ways to regulate protein activity (including enzyme activity):1. allosteric control2. multiple forms of enzymes (isozymes)3. reversible covalent modification4. interaction with regulatory proteins5. irreversible covalent modification, including proteolytic activationKey Concepts, continued1. Allosteric control– conformational changes• 2 conformations in equilibrium, "R" (more active) & "T" (less active)– allosteric activators (positive effectors/modulators)– allosteric inhibitors (negative effectors/modulators)• often feedback inhibitors (product of pathway inhibits firstcommitted step in pathway)• Allosterically regulated enzymes always multi-subunit• Aspartate transcarbamoylase (ATCase) as an example– homotropic effector = activator (substrate aspartate)– heterotropic effectors (activator = ATP; inhibitor = CTP)2. Isozymes– Multiple forms of an enzyme that catalyze the same reaction– Different kinetic parameters like Km, and/or different allostericregulation, with physiological consequences– Hexokinase -- different forms in liver vs. muscle reflect the differentroles of those tissues in the body.Learning Objectives• Terminology (some are review): quaternary structure, multimeric protein,homopolymeric protein, heteropolymeric protein, ligand, binding site,fractional saturation, feedback inhibition, cooperativity, cooperativebinding, allosteric, homotropic effector/regulator, heterotropiceffector/regulator, allosteric activator (positive heterotropiceffector/regulator), allosteric inhibitor (negative heterotropiceffector/regulator), protomer, prosthetic group, isozyme.• Briefly explain the allosteric regulation of ATCase, including its quaternarystructure, its role in metabolism, and how its activity is regulated byallosteric inhibition and activation. Include the physiological rationale forthe inhibition and activation.• Sketch plots of Vo vs. [S] for an allosteric enzyme that illustrate positivehomotropic regulation and positive and negative heterotropic regulation,with ATCase as an example. Specifically, sketch (all on the same axes)for ATCase: Vo vs. [aspartate] curves with no heterotropic regulatorspresent, with an allosteric inhibitor present, and with an allosteric activatorpresent.• Explain the biological usefulness of isozymes, and discuss the example ofmuscle hexokinase vs. liver glucokinase in terms of difference in functionof the tissues.Regulatory enzymesIn general,• Catalyze essentially irreversible metabolic reactions (ΔG’ large, neg.)and/or• Catalyze the first committed step in a metabolic pathway• Regulation of such steps permits efficient regulation of flux ofmetabolites through just that pathway⊗⊗• "Committed step": As a result of this step, metabolite (small molecule) is committed to continue down that pathway to endproduct.• No other branches lead to different endproducts that need to be regulated separately.• FIRST committed step = the most efficient step for regulation of the rate -- that should also be the slowest step in pathway, controlling "flow" of matter to endproduct whose concentration you want to regulate.5 principal ways protein/enzyme activity is regulated1. Allosteric control• Regulation of binding affinity for ligands, and/or of catalytic activity, byconformational changes caused by binding of the same or otherligands at other sites on protein ("allosteric effects")• Changes involve simple association/dissociation of small molecules, soenzyme can cycle rapidly between active and inactive (or more and lessactive) states.3. Multiple forms of enzymes (isozymes)• Isozymes (isoenzymes) = multiple forms of enzyme that catalyze same reaction but are products of different genes (so different amino acid sequences)• Isozymes differ slightly in structure, and kinetic and regulatory properties are different.• Can be expressed in different tissues or organelles, at different stages of development, etc.2. Interaction with regulatory proteins • Binding of a different protein to the enzyme alters the enzyme activity (activates or inhibits the enzyme), usually by causing conformational change.BIOC 460, Spring 2008LEC 15, Enzymes - Regulation 1 25 principal ways protein/enzyme activity is regulated4. Reversible covalent modification• Modification 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, soenzyme can cycle between active and inactive (or more and less active)states.5. Proteolytic activation• Irreversible 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-binding specific inhibitory protein inactivates it.Allosteric Regulation• Multisubunit enzymes (more than one active site per enzyme)• Regulation of binding affinity for ligands (like substrates) and/orcatalytic activity (kcat)• Conformational changes linked with ligand binding– homotropic effects: binding of "primary" ligand (substrate for anenzyme, O2 for hemoglobin, etc.) can alter affinity of other bindingsites on molecule for that same ligand– heterotropic effects: binding of other ligands (regulatory signalingmolecules), to different sites from the primary ligand ("regulatorysites") can cause conformational changes that alter primary ligandbinding affinity or catalytic activity– Sometimes regulatory sites are on different subunits
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