BIOC 460, Spring 2008LEC 16-17, Enzymes - Regulation 2-3 1Lectures 16-17Enzymes: Regulation 2-3Reversible covalent modificationAssociation with regulatory proteinsIrreversible covalent modification/proteolytic cleavageReading: Berg, Tymoczko & Stryer, 6th ed.,Chapter 10, pp. 283-299, Chapter 14, pp. 389-391Problems: pp. 300-302, Chapter 10: #7, 10, 12, 13Key Concepts• 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 modification -- example:• phosphorylation/dephosphorylation• phosphorylation (phosphoryl transfer from ATP to specific -OHgroup(s) on protein) catalyzed by protein kinases• dephosphorylation (hydrolytic removal of the phosphate groups)catalyzed by protein phosphatases4. interaction with regulatory proteins – examples:• protein kinase A (PKA)• Ca2+-calmodulin-dependent kinases5. irreversible covalent modification, including proteolytic activation (zymogen activation)• examples:– digestive proteases like chymotrypsin and trypsin– blood clotting cascadeLearning Objectives• Terminology: cAMP, consensus sequence, pseudosubstrate, cascade,reciprocal regulation, zymogen• Describe in general terms how cells carry out reversible covalentmodification of enzymes, and how the modification would be removed.• Name (“generic” names) the types of enzymes that catalyzephosphorylation and dephosphorylation of proteins, specify what types ofamino acid functional groups are generally the targets of phosphorylation,and show the structure of such an enzyme functional group before andafter phosphorylation.• Explain whether the dephosphorylation reaction is actually the chemicalreverse of the phosphorylation reaction, and if not, what type of reactionthe dephosphorylation represents.• Explain the regulation of protein kinase A (PKA) activity by cAMP,including quaternary structural changes in PKA triggered by cAMPbinding. How does the term "pseudosubstrate" relate to the role of theregulatory subunits in PKA?• What are the 2 enzymes involved in glycogen metabolism whose activitiesare reciprocally regulated by phosphorylation/dephosphorylation?• Briefly discuss the structure of calmodulin (± Ca2+), including structure ofthe “EF hand” motif, and how Ca2+-calmodulin activates target proteins asan example of how a regulatory protein works.Learning Objectives, continued• Describe the general mechanism by which zymogens are activated →active enzymes.• Briefly describe the structural change that occurs upon the activation ofchymotrypsinogen, including what changes occur in the active site.• Discuss the protective mechanism that keeps prematurely activatedpancreatic digestive enzymes inside the acinar cells from autodigestingthe pancreas, and describe/name an example.• Give examples of protease inhibitors that inhibit a) elastase, and b)thrombin.• Explain how a deficiency (or an inactivating chemical event) in α1-antiproteinase (formerly called α1-antitrypsin) contributes to emphysema.• Explain how a cascade of catalysts (e.g., in PKA activation, or in bloodclotting) results in amplification of a signal.• Outline (no specific factor names/numbers needed) how the intrinsic andextrinsic pathways converge in the final common pathway in bloodclotting, including roles of prothrombin, thrombin, fibrinogen andfibrin. (You don’t have to learn specific names/numbers of clotting factorsin intrinsic and extrinsic pathways.)• Name the type (class) of enzyme represented by most of the clottingfactors.Learning Objectives, continued• Describe the covalent modification of some of the clotting factors thatrequires the participation of vitamin K. What is the structure of themodified amino acid R group, and what is the 3-letter abbreviation of themodified residue? What ion is bound by the modified amino acidresidues?• Briefly discuss how clots are confined to the area of injury, including therole of heparin and antithrombin III.• Describe how fibrinolysis (clot breakdown) is achieved, with names offactors involved. What is TPA?3. Reversible Covalent Modification• Modification of catalytic or other properties of proteins by covalentattachment of a modifying group• modification reaction catalyzed by a specific enzyme.• modifying group removed by catalytic activity of a different enzyme• Enzyme can cycle between active and inactive (or more and less active)states.• allosteric regulation: "instant" sensing of local concentration signals, so rapid activity changes• covalent modifications: generally cause slower and longer-lasting effects than from allosteric regulation, with coordinated systemic effects (e.g., a hormone can trigger covalent modification events that change activities of metabolic enzymes in a variety of tissues and many cells.)• Activities of modifying/demodifying enzymes themselves are regulated, allosterically (making process sensitive to changes in concentration of small molecules that act as "signals"), or by another reversible covalent modification process, or both.BIOC 460, Spring 2008LEC 16-17, Enzymes - Regulation 2-3 2• Histone acetylation/deacetylation– Histones: eukaryotic proteins in chromatin– involved in DNA packaging andregulation of gene expression– very high percentages of Lys & Arg residues– heavily acetylated histones associated with actively transcribed genes– acetylation of Lys residues catalyzed by specific acetyl transferases– removal of the acetyl groups catalyzed by deacetylases– modifying/demodifying enzymes (acetylases and deacetylases)themselves regulated by phosphorylation/dephosphorylationPhosphorylation/dephosphorylation• probably the most common means of regulating enzymes, membranechannels, virtually every metabolic process in eukaryotic cells• Phosphorylation– Kinase: Any enzyme catalyzing phosphoryl transfer involving ATP orother nucleoside triphosphate• named for molecule that "receives" phosphate group• e.g., hexokinase transfers terminal phosphate from ATP to a varietyof hexose sugars like glucose (→ glucose-6-phosphate).– General reaction catalyzed by kinases:– (target) R-OH + ATP <==> R-OPO32– + ADP– Product =
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