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UIUC MCB 450 - 450 F15 Lect 12 for posting

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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 44MCB 450Lecture 12Catalytic strategies used by enzymesInhibition of enzyme activityExample of an enzyme mechanism:peptide hydrolysis by chymotrypsin12-11. Covalent catalysis: the active site contains a reactive group (nucleophile) that is briefly covalently modified. 2. General acid-base catalysis: a molecule other than water donates or accepts a proton.3. Metal ion catalysis: metal ions can function in various ways, e.g…stabilize a –ve charge on a reaction intermediategenerate a nucleophile by deprotonating waterbind to S, increase interactions with E, increasing binding energy4. Catalysis by approximation & orientation: Enzyme brings two substrates close together and orients the reacting parts of substratemolecules for reactionCatalytic strategies used by enzymes12-2nucleophilic attack by a group on E on acarbonyl carbonE is covalently linked to the substrate = acyl enzyme intermediate SubstrateCatalysis through formation of covalent bonds between E and SCovalent catalysis12-3Nucleophiles:Functional groups rich in e-that can combine with, and giveup e- to, e- -deficient electrophiles- Acid-base catalysis: in which acceleration of a reaction is achieved by catalytic transfer of a proton- Enzymes use the side chains of  that can donate or accept protons under the nearly neutral pH conditions in cells!General acid-base catalysis12-4The H+ or OH- participating in the reaction is created in the transition state by another molecule or a group on the enzyme:Imidazole acts as "general base"and "abstracts" proton from water, creating nucleophilic OH-Amino acids involved in acid-base catalysisSer-OH v. weakly acidic,but can be deprotonatedby e.g. an adjacent basicHis in an active site12-5Water molecule activated to serve as nucleophile: attacks carbonyl carbon of peptide bondZinc in the metalloprotease thermolysin 12-6Catalysis by approximation & orientation 12-7 Bond Enzyme (E)Effect of heat on enzyme activity12-8HEAT INCREASES THERMAL MOTION,MAKING E-S INTERACTIONS MORE LIKELYDENATURATION, LOSSOF 3-D STRUCTUREAbility of an enzyme to use the side chains of  that can donate or accept protonsmeans that………..Enzyme activity is reponsive to pH, and that enzymes have a pH optimum……..at which the greatest # of enzyme molecules are in theappropriate state of protonation/deprotonation….12-9Effect of pH on enzyme activity12-10Measure enzyme activity starting at pH 2,then at increasing pH up to pH 11Activity goes up as criticallow pKa group on 2is deprotonatedActivity then goes down as criticalhigher pKa group on 1is deprotonatedInterpretation….TWO CRITICAL IN ACTIVE SITE12-11Inhibition of enzyme activityEnzymes can be inhibited by specific small(ish) molecules that interfere withcatalysis and slow or halt a reaction, e.g. pharmaceutical agents and toxins1. Reversible: EI complex can dissociate rapidly.Different kinds: can be distinguished kinetically2. Irreversible: inhibitor becomes tightly bound to enzyme -covalently or non-covalentlyEI complex dissociates very s l o w l y, if at all12-12Have different effects on Km and Vmax,which we can think of as as a "tug-of-war"between dissociation constants for I and S binding to E Types of reversible inhibition12-132. Uncompetitive: inhibitor binds to separate site onenzyme, but only to ES complex3. Noncompetitive (“pure”): inhibitor binds to separatesite on E or ES complex1. Competitive: inhibitor binds to enzyme's active siteCompetitive inhibitors- Often resemble substrate structurally- Compete with substrate for active site- While in active site, prevent binding of substrate- Combine with E to form complex, but do not lead to catalysis- Combination of E + I lowers efficiency of enzyme- Competitive inhibition can be analyzed quantitatively bysteady-state kinetics12-14“TRANSITION STATE”SICompetitive inhibition12-15KmKmappKmappEffect of a competitiveinhibitor on Km and Vmax12-16Effect of a competitive inhibitor on Km and Vmax12-17Examples of competitive inhibitors- of hydroxymethylglutaryl coenzyme A reductase- block cholesterol synthesisS-CoAO12-18Uncompetitive inhibition12-19Effect of uncompetitive inhibitor on Km and VmaxKm and Vmax reduced by equivalent amountsIn UNcompetitive inhibition,I binding pulls equilibrium towards ESIand therefore favors ES formation,i.e. as ES disappears, more E + S associateto restore the equilibrium amount of ES.Increased tendency of S to associate with Emeans Km .12-2012-21 Noncompetitive inhibitionORIn NON-competitive inhibition,ki and ki’ are equal and cancel outone another’s effect on Km,although Vmax goes down because active Emolecules are being removedfrom the reaction mix.Effect of noncompetitive inhibitor on Km and Vmax12-22The inhibitor I inhibits the reaction S  P, catalyzed by the enzyme E. Measurement of initial velocities of the reaction, in presence and absence of the inhibitor yielded the following data:V0 [S] [I](M min-1) (M ) (M) 0.2 0.005 00.222 0.0067 00.242 0.01 00.274 0.02 0---------------------------------------------------------------------------------------------V0 [S] [I](M min-1) (M) (M) 0.162 0.005 0.010.185 0.0067 0.010.216 0.01 0.01 0.254 0.02 0.01 a. What is the nature of inhibition by inhibitor I when S is the substrate ? b. What is the value of the inhibition constant Ki ?Solving problems on enzyme inhibition12-23Solving problems on enzyme inhibition- Calculate 1/V0 and 1/[S]- Plot the double reciprocal plot:1/V0 on the y-axis and 1/[S] on the x-axis- Look at the straight lines in absence of I and in presence of I- Diagnose type of inhibition- The graph also gives you the values of Kmapp12-24NOT OTHER SERINES IN THE PROTEIN12-25REACTIVE AMINO ACID IN ACTIVE SITE ISCOVALENTLY MODIFIED BY AGROUP-SPECIFIC REAGENT12-26DIPF = acetylcholinesterase inhibitorDIPFBlocks normal deactivation of neurotransmitter


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