PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 4813-1MCB 450Lecture 13Enzyme Regulation: AllosteryConformational Changes in Hemoglobin upon O2-bindingOther Ways to Regulate EnzymesEXAM 2 Thursday, Oct 15On material covered in Lectures 8 through 13REVIEW FOR EXAM 2Thursday Oct 15HERE, instead of lectureIF YOU WOULD LIKE ME TO GO OVER A PARTICULAR TOPIC,PLEASE E-MAIL ME BY WEDNESDAY Oct 14, 2 PMRegulation of enzyme activity1. Reversible, non-covalent binding of a regulatory moleculeto a second site on enzyme = allosteric modulator/effector2. Reversible, covalent modification (esp. phosphorylation)3. Proteolytic cleavage to remove a segment of a peptideGeneral effects are to alter… conformationsubstrate bindingcatalytic activityBy….NOT THE ACTIVE SITE13-2http://www.biocarta.com/pathfiles/isoleucinePathway.aspMany enzymes act in sequences in biosynthetic pathways13-3Issue: 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 Ilethrough a succession of biosynthetic intermediatesRegulatory step in many biosynthetic pathwaysis catalyzed by an allosteric enzyme1. One enzyme in pathway sets rate of overall sequence because it catalyzes the slowest or rate-limiting step2. In a multienzyme pathway, it's best if the first enzyme is regulated3. The first, regulatory enzyme is specifically inhibited by the end-product of the pathway4. End-product binds to enzyme, but at a site other than the active site, and the first reaction is slowed.END-PRODUCT ORFEEDBACKINHIBITIONOF FIRSTENZYME INPATHWAYMulti-enzyme pathwaycatalyzes stepwiseconversion of Thr to Ilethrough a succession ofbiosynthetic intermediates13-4Allosteric effectors• Can be inhibitors: often the end-product• Can be activators: often the substrate itselfRegulatory 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 proteins13-5V versus [S] plot for a homotropic enzymewhose substrate is an allosteric activator13-6KmSIGMOIDAL PLOT INDICATES THAT BINDINGOF ONE MOLECULE OF S ENHANCES BINDINGOF ANOTHER….….. THIS IS CALLED "COOPERATIVITY"How do we get cooperativity?(in a homotropic allosteric enzyme)13-71. Binding of S to one active site affects properties of otheractive sites in the same enzyme, e.g. by facilitating S binding2. Cannot be obtained with a single-subunit protein with asingle substrate binding siteb/c each S molecule binds independently, & cannot affectbinding of S to another enzyme molecule3. So, allosteric enzyme most likely hasmultiple (≥ 2) active sitesmultiple (≥ 2) subunits/domainsMost allosteric proteins have 2 or more subunits13-8How do we explain allosteric regulation?Threonine dehydratase: dimer of dimers13-9How does this explain the sigmoidal plot?Allosteric interactions: the “concerted” model 13-10MONOD,WYMAN,& CHANGEUXAssumptions.….. (for positive cooperativity):1. Multi-subunit proteins2. Each protein has two conformational states:“T” = less active“TENSE / STIFF / INACTIVE”“R” = high affinity “RELAXED / READY FOR EXCERCISE / LIMBERED UP” 6. S binds much more readily to R form than to T-form. 3. T and R are in equilibrium, with T being the more stable,hence more common state4. Spontaneous conversion of a T subunit to R possible,& remaining Ts convert too5. Concerted model requires all subunits to be either in T or in R state(no hybrids allowed, = symmetry rule)The “concerted” model 13-11[T] >>> [R]: MOST ENZYMEMOLECULES IN T-FORM,WHICH CAN HARDLY BIND S, SOLITTLE ACTIVITY AT LOW [S] AS [S] IS RAISED, THERE WILL EVENTUALLYBE ENOUGH S PRESENT SO THAT WHEN ANR-FORM OF THE ENZYME APPEARS, S WILLBIND IT, AND ALL SUBUNITS WILL BETRAPPED IN THE R-FORM AND ALSO ABLETO BIND SAS MORE ENZYMES ARE TRAPPED INTHE R-FORM, S IS LESS LIKELY TOCOLLIDE UNPRODUCTIVELY WITHT-FORM, AND INCREASINGLY LIKELYTO ENCOUNTER R-FORMS BINDING OF S TO THE R-FORM BECOMESEASIER AS MORE AND MORE ENZYME ISIN 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 V013-12Allosteric activators vs inhibitors ACTIVATOR OR S SHIFTST <-> R EQUILIBRIUMTOWARDS RALLOSTERIC INHIBITORSHIFTS T <-> R EQUILIBRIUMTOWARDS T13-13ATP STABILIZES THE R-FORM,MAKING IT EASIER FOR S TO BIND,i.e. LOWERING THE THRESHOLDCONCENTRATION OF S NEEDEDFOR ACTIVITYEffects of regulators on the allosteric enzymeaspartate transcarbamoylase13-14CTP STABILIZES THE T-FORM,MAKING IT MORE DIFFICULT FORS BINDING TO CONVERT THEENZYME TO THE R-FORM,Heterotropic versus homotropic13-15• Regulatory enzymes whose substrate and regulator are…..- different molecules = heterotropic:disruption of T<->R equilibrium by regulators - the same = homotropicdisruption of T<->R equilibrium by substratesBecause curves aren't hyperbolic,the protein is not obeying Michaelis-Menten kinetics, so, although we canfind 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 modulatorsare distinct from those ofuncompetitive & non-competitive inhibitorsEffects of +ve and -ve allosteric modulators on “Km“i.e. on response of V0 to [S]: 13-16Activator (+) lowers “K0.5”(usually without changingVmax), and increases V0for any value of [S]Inhibitor (-) raises “K0.5”13-17ACTIVITY OF AN ALLOSTERIC ENZYMEIS MORE SENSITIVE TO CHANGES IN[S] NEAR Km THAN AN M-M ENZYME WITHTHE SAME Km AND VmaxKmAdvantage 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 neighboringsubunits that increase their affinity for substrate,which they then bind more readily, andthen 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
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