Berg Tymoczko Stryer Biochemistry Seventh Edition Chapter 10 Regulatory Strategies Copyright 2012 by W H Freeman and Company Concepts proteins include Beyond controlling the amount of enzyme e g transcription levels protein degradation strategies employed to regulate the activity of enzymes and other cid 190 Activity regulatory mechanisms Allosteric control Isozymes Reversible covalent modifications de phosphorylation Proteolytic activity zymogens Examples of regulatory strategies cid 190 Aspartate transcarbamoylase cid 190 Protein Kinase A Cooperativity and R T equilibrium CTP feedback inhibition ATP activation Allosteric regulation of covalent modification cAMP and pseudosubstrate Amplification Regulations at key points along enzymatic pathways Enzymes that are subjected to tight regulation often catalyze committed steps along specific metabolic pathway A committed catalytic step is basically irreversible The product of a committed step will essentially continue down the pathway to final product Targeting the first committed catalytic step along a enzymatic pathway helps regulate the endproduct formation without unnecessary loss of energy Enz1 A B Enz2 x Enz3 C Enz6 x Feedback regulation inhibition or activation Enz4 D E Enz5 F final product 1 L Enz7 M Enz8 N O Enz9 Enz10 P final product 2 Feedback regulation Allosteric controls Enzymes regulated by allosteric control allosteric enzymes are multi subunits proteins Regulation of catalytic kcat and binding activities by inducing significant conformational changes effected by the substrate it self at catalytic sites cooperativity between multiple catalytic subunits Homotropic effects By other ligands binding at sites distinct from the catalytic site to increase or decrease enzymatic activity Heterotropic effects Heterotropic effectors increase catalytic rate Heterotropic inhibitors decrease catalytic rate Allosteric controls often involved in rapid switching between a highly active catalytic state R and a less active state T of the enzyme Useful for quick responses to rapid metabolic changes and needs Allosteric controls continued Allosteric enzymes do not follow Michaelis Menten kinetics Cooperative binding binding of 1 substrate lead to increased binding affinity for substrate and higher catalytic activity at other active sites on an allosteric enzyme Non cooperative enzyme Allosteric enzyme High S High binding affinity for substrate and high catalytic rates Increase in S lead to a sharp increase in catalytic rate high slope until maximum rate is reached Low S low binding affinity for substrate and low catalytic rates Aspartate Transcarbamoylase is allosterically inhibited ATCase allosteric enzyme involved in a committed step step 2 of a multisteps enzymatic pathway leading to the biosynthesis of pyrimidines nucleotides building block of nucleic acid role in enzyme activity ATCase committed step The end product CTP controls ATCase catalytic rates by negative feedback heterotropic effects Understanding ATCase catalysis from its structure ATCase has 12 subunits 6 catalytic chains organized in 2 trimers 3 active sites trimers 6 regulatory chains organized in 3 dimers Many contacts between regulatory dimer and catalytic trimers Understanding ATCase catalysis from its structure To understand the cooperative effects of carbamoyl phosphate and aspartate substrates on the ATCase a bisubstrate analog PALA was synthesized Notice the close resemble of PALA with reaction intermediate Understanding ATCase catalysis from its structure Binding of PALA to catalytic sites of ATCase large conformational changes of ATCase T state of ATCase tense compact form with low catalytic activity R state of ATCase relaxed expanded form with high catalytic activity PALA and so substrates changes equilibrium between T and R states toward the R state Cooperative effects Low S Tstate Rstate lower binding affinity for S low activity High S Rstate Tstate higher binding affinity for S high activity Steep rate increase within narrow changes in S due to conformational change Feedback inhibition by CTP CTP acts by modulating the equilibrium between ATCase T and R states CTP binds to regulatory subunits preferentially in T state CTP stabilizes the low catalytic efficiency T state CTP shift the equilibrium toward the T state CTP decrease the affinity of ATCase for carbamoyl phosphate and aspartate Feedback inhibition by CTP In terms of kinetics CTP make it more difficult for bound substrates to convert ATCase to the R state and thus make it more difficult to produce more CTP Need more substrate to achieve the same catalytic rate somewhat equivalent to an increase in apparent KM Feedback inhibition leads to reduce rate of production of CTP by ATCase as a response to high CTP ATP is an allosteric effector of ATCase Under high ATP the kinetic of ATCase show less pronounced sigmoidal behavior ATP competes with CTP binding on regulatory subunits High ATP prevent ATCase inhibition by CTP But catalytic rates increase slightly with ATP indicates that ATP favors the R state of ATCase ATP favors pyrimidine synthesis to balance purine to pyrimidine ratios in cells High ATP energy available signals that conditions are good to prepare for mRNA synthesis or DNA replication Regulatory strategy by isozymes Isozymes multiple form of enzyme catalyzing the same reaction in different manners and or in different tissues Differences between isozymes include different KM and different sensitivity to regulators Isozymes are encoded by different genes in different tissues Isozymes allow fine tuning of metabolic process to meet the needs of specific tissues or development stages use of isozymes often involved in longer term regulations rather than fast responses to metabolic changes Rat heart LDH profile Expression profile of 2 Lactate dehydrogenase LDH isozymes during development M isozyme anaerobic H isozyme aerobic Combinations of isozymic polypeptides subunits blue and red provide modulatory responses to the allosteric inhibitor pyruvate Regulation of enzyme activity by covalent modifications Extremely common strategy to modify enzymes and protein activity Covalent modifications are often reversible and are done by enzymes which are themselves subjected to regulation Phosphorylated serine Phosphorylation and dephosphorylation modifications Phosphorylation Reaction catalyzed by enzymes called kinases Transfer of a phosphoryl group from ATP to residues in a protein Ser OH Thr OH Tyr OH