13 1 MCB 450 Lecture 13 Enzyme Regulation Allostery Conformational Changes in Hemoglobin upon O2 binding Other Ways to Regulate Enzymes EXAM 2 Thursday March 12 On material covered in Lectures 8 through 13 REVIEW FOR EXAM 2 Thursday March 12 HERE instead of lecture IF YOU WOULD LIKE ME TO GO OVER A PARTICULAR TOPIC PLEASE E MAIL ME BY MONDAY March 11 2 PM 13 2 Regulation of enzyme activity By 1 Reversible non covalent binding of a regulatory molecule to a second site on enzyme allosteric modulator effector NOT THE ACTIVE SITE 2 Reversible covalent modification esp phosphorylation 3 Proteolytic cleavage to remove a segment of a peptide General effects are to alter conformation substrate binding catalytic activity 13 3 Many enzymes act in sequences in biosynthetic pathways Example a multi enzyme pathway catalyzes stepwise conversion of Thr to Ile through a succession of biosynthetic intermediates Issue What if the cell has enough Ile How can it down regulate the pathway http www biocarta com pathfiles isoleucinePathway asp 13 4 Regulatory step in many biosynthetic pathways is catalyzed by an allosteric enzyme 1 One enzyme in pathway sets rate of overall sequence because it catalyzes the slowest or rate limiting step END PRODUCT 2 In a multienzyme pathway it s best OR if the first enzyme is regulated FEEDBACK INHIBITION OF FIRST 3 The first regulatory enzyme is ENZYME IN specifically inhibited by the PATHWAY end product of the pathway 4 End product binds to enzyme but at a site other than the active site and the first reaction is slowed Multi enzyme pathway catalyzes stepwise conversion of Thr to Ile through a succession of biosynthetic intermediates 13 5 Allosteric effectors Can be inhibitors often the end product Can be activators often the substrate itself Regulatory enzymes whose substrate and regulator are different molecules heterotropic the same homotropic Allosteric proteins Do not conform to Michaelis Menten kinetics seen from plot of V0 vs S 13 6 V versus S plot for a homotropic enzyme whose substrate is an allosteric activator Km SIGMOIDAL PLOT INDICATES THAT BINDING OF ONE MOLECULE OF S ENHANCES BINDING OF ANOTHER THIS IS CALLED COOPERATIVITY 13 7 How do we get cooperativity in a homotropic allosteric enzyme Binding of S to one active site affects properties of other active sites in the same enzyme e g by facilitating S binding Cannot be obtained with a single subunit protein with a single substrate binding site b c each S molecule binds independently cannot affect binding of S to another enzyme molecule So allosteric enzyme most likely has multiple 2 active sites multiple 2 subunits domains 13 8 Most allosteric proteins have 2 or more subunits 13 9 Threonine dehydratase dimer of dimers How do we explain allosteric regulation 13 10 Allosteric interactions the concerted model Assumptions for positive cooperativity MONOD WYMAN CHANGEUX 1 Multi subunit proteins 2 Each subunit has two conformational states T less active TENSE STIFF INACTIVE R high affinity RELAXED READY FOR EXCERCISE LIMBERED UP 3 T and R are in equilibrium with T being the more stable hence more common state although spontaneous conversion to R possible 4 Concerted model requires all subunits to be either in T or in R state no hybrids 5 S binds much more readily to R form than to T form 6 T R transition of subunit induced upon binding of ligand substrate or activator 7 Binding of ligand switches conformation of all subunits i e induces the T R transition in neighboring subunits How does this explain the sigmoidal plot 13 11 The concerted model T R MOST ENZYME MOLECULES IN T FORM WHICH CAN HARDLY BIND S SO LITTLE ACTIVITY AT LOW S AS S IS RAISED THERE WILL EVENTUALLY BE ENOUGH S PRESENT THAT WHEN AN R FORM OF THE ENZYME APPEARS S WILL BIND IT AND FLIP ALL SUBUNITS TO R FORM TRAPPING IN THE ACTIVE FORM 13 12 The concerted model AS MORE ENZYMES ARE TRAPPED IN BINDING OF S TO THE R FORM BECOMES THE R FORM S IS LESS LIKELY TO EASIER AS MORE AND MORE ENZYME IS COLLIDE UNPRODUCTIVELY WITH IN THE R FORM AND THE MORE S BOUND THE MORE ACTIVITY R R T FORM AND INCREASINGLY LIKELY TO ENCOUNTER R FORMS BINDING OF S SHIFTS THE T R EQUILIBRIUM IN FAVOR OF R ACCOUNTING FOR THE SHARP INCREASE IN V0 13 13 Allosteric activators vs inhibitors ALLOSTERIC INHIBITOR SHIFTS T R EQUILIBRIUM TOWARDS T ACTIVATOR OR S SHIFTS T R EQUILIBRIUM TOWARDS R 13 14 Effects of regulators on the allosteric enzyme aspartate transcarbamoylase ATP STABILIZES THE R FORM MAKING IT EASIER FOR S TO BIND i e LOWERING THE THRESHOLD CONCENTRATION OF S NEEDED FOR ACTIVITY CTP STABILIZES THE T FORM MAKING IT MORE DIFFICULT FOR S BINDING TO CONVERT THE ENZYME TO THE R FORM 13 15 Heterotropic versus homotropic Regulatory enzymes whose substrate and regulator are different molecules heterotropic disruption of T R equilibrium by regulators the same homotropic disruption of T R equilibrium by substrates 13 16 Effects of ve and ve allosteric modulators on Km i e on response of V0 to S Activator lowers K0 5 usually without changing Vmax and increases V0 for any value of S Inhibitor raises K0 5 Because curves aren t hyperbolic the protein is not obeying MichaelisMenten kinetics so although we can find a value of S where V0 0 5 Vmax we shouldn t use the term Km but instead K0 5 Kinetic effects of ve allosteric modulators are distinct from those of uncompetitive noncompetitive inhibitors 13 17 Km Advantage of allosteric regulation ACTIVITY OF AN ALLOSTERIC ENZYME IS MORE SENSITIVE TO CHANGES IN S NEAR Km THAN AN M M ENZYME WITH THE SAME Km AND Vmax 13 18 Allosteric interactions sequential model KOSHLAND T 1 Binding of S switches conformation of subunit to which it s bound but does not induce the T R transition in neighboring subunits 2 However T R in one subunit does induce changes in neighboring subunits that increase their affinity for substrate which they then bind more readily and then undergo T R themselves i e T subunit with an R neighbor has higher affinity for ligand than T subunit with a T neighbor 3 Rate constants for S binding K1 K2 K3 K4 get increasingly favorable as more and S binds 4 Sequential model better at explaining negative cooperativity in which binding of one S decreases affinity of other sites for S ALLOSTERIC ENZYMES MAY BEHAVE ACCORDING TO SOME COMBINATION OF CONCERTED SEQUENTIAL MODELS 13 19 O2 binding by hemoglobin an allosteric protein BIG PICTURE Hemoglobin is a tetramer myoglobin a monomer Hemoglobin and myoglobin bind O2 on heme groups
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