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 PMRegulation of enzyme activity 1. Reversible, non-covalent binding of a regulatory molecule to a second site on enzyme = allosteric modulator/effector 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 By…. NOT THE ACTIVE SITE 13-2http://www.biocarta.com/pathfiles/isoleucinePathway.asp Many enzymes act in sequences in biosynthetic pathways 13-3 Issue: What if the cell has enough Ile? How can it "down-regulate" the pathway? Example: a multi-enzyme pathway catalyzes stepwise conversion of Thr to Ile through a succession of biosynthetic intermediatesRegulatory 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 2. In a multienzyme pathway, it's best if the first enzyme is regulated 3. The first, regulatory enzyme is specifically inhibited by the 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. END-PRODUCT OR FEEDBACK INHIBITION OF FIRST ENZYME IN PATHWAY Multi-enzyme pathway catalyzes stepwise conversion of Thr to Ile through a succession of biosynthetic intermediates 13-4Allosteric 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 • Do not conform to Michaelis-Menten kinetics - seen from plot of V0 vs [S]...... Allosteric proteins!13-5V versus [S] plot for a homotropic enzyme!!whose substrate is an allosteric activator!13-6 Km SIGMOIDAL PLOT INDICATES THAT BINDING OF ONE MOLECULE OF S ENHANCES BINDING OF ANOTHER…. ….. THIS IS CALLED "COOPERATIVITY"How do we get cooperativity? (in a homotropic allosteric enzyme) 13-7 1. Binding of S to one active site affects properties of other active sites in the same enzyme, e.g. by facilitating S binding 2. 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 3. So, allosteric enzyme most likely has multiple (≥ 2) active sites multiple (≥ 2) subunits/domainsMost allosteric proteins have 2 or more subunits 13-8How do we explain allosteric regulation? Threonine dehydratase: dimer of dimers 13-9Assumptions.….. (for positive cooperativity): 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.. Allosteric interactions: the “concerted” model 13-10 MONOD, WYMAN, & CHANGEUX How does this explain the sigmoidal plot?The “concerted” model 13-11 [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 FORMAS MORE ENZYMES ARE TRAPPED IN THE R-FORM, S IS LESS LIKELY TO COLLIDE UNPRODUCTIVELY WITH T-FORM, AND INCREASINGLY LIKELY TO ENCOUNTER R-FORMS BINDING OF S TO THE R-FORM BECOMES EASIER AS MORE AND MORE ENZYME IS IN THE R-FORM, AND THE MORE S BOUND, THE MORE ACTIVITY. [R] >>> [R] The “concerted” model BINDING OF S SHIFTS THE T <-> R EQUILIBRIUM IN FAVOR OF R, ACCOUNTING FOR THE SHARP INCREASE IN V0 13-12Allosteric activators vs inhibitors ACTIVATOR OR S SHIFTS T <-> R EQUILIBRIUM TOWARDS R ALLOSTERIC INHIBITOR SHIFTS T <-> R EQUILIBRIUM TOWARDS T 13-13ATP STABILIZES THE R-FORM, MAKING IT EASIER FOR S TO BIND, i.e. LOWERING THE THRESHOLD CONCENTRATION OF S NEEDED FOR ACTIVITY Effects of regulators on the allosteric enzyme aspartate transcarbamoylase 13-14 CTP STABILIZES THE T-FORM, MAKING IT MORE DIFFICULT FOR S BINDING TO CONVERT THE ENZYME TO THE R-FORM,Heterotropic versus homotropic 13-15 • 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 substratesBecause curves aren't hyperbolic, the protein is not obeying Michaelis- Menten 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 & non- competitive inhibitors Effects of +ve and -ve allosteric modulators on “Km“ i.e. on response of V0 to [S]: "13-16 Activator (+) lowers “K0.5” (usually without changing Vmax), and increases V0 for any value of [S] Inhibitor (-) raises “K0.5”13-17 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 Km Advantage of allosteric regulation......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
View Full Document
Unlocking...