MCB 450 Lecture 12 Catalytic strategies used by enzymes Inhibition of enzyme activity Example of an enzyme mechanism: peptide hydrolysis by chymotrypsin 12-11. 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… ü stabilize a –ve charge on a reaction intermediate ü generate a nucleophile by deprotonating water ü bind to S, increase interactions with E, increasing binding energy 4. Catalysis by approximation & orientation: Enzyme brings two substrates close together and orients the reacting parts of substrate molecules for reaction Catalytic strategies used by enzymes 12-2nucleophilic attack by a group on E on a carbonyl carbon E is covalently linked to the substrate = acyl enzyme intermediate Substrate Catalysis through formation of covalent bonds between E and S Covalent catalysis 12-3 Nucleophiles: Functional groups rich in e- that can combine with, and give up 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 αα that can donate or accept protons under the nearly neutral pH conditions in cells ! General acid-base catalysis 12-4 The 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 catalysis Ser-OH v. weakly acidic, but can be deprotonated by e.g. an adjacent basic His in an active site 12-5Water molecule activated to serve as nucleophile: attacks carbonyl carbon of peptide bond Zinc in the metalloprotease thermolysin 12-6Catalysis by approximation & orientation 12-7 !!!Bond!!!!!Enzyme!(E)!Effect of heat on enzyme activity 12-8Ability of an enzyme to use the side chains of αα that can donate or accept protons means that……….. Enzyme activity is reponsive to pH, and that enzymes have a pH optimum ……..at which the greatest # of enzyme molecules are in the appropriate state of protonation/deprotonation…. 12-9 Effect of pH on enzyme activity12-10"Measure enzyme activity starting at pH 2, then at increasing pH up to pH 11 Activity goes up as critical low pKa group on αα2 is deprotonated Activity then goes down as critical higher pKa group on αα1 is deprotonatedInterpretation…. TWO CRITICAL αα IN ACTIVE SITE 12-11Inhibition of enzyme activity Enzymes can be inhibited by specific small(ish) molecules that interfere with catalysis and slow or halt a reaction, e.g. pharmaceutical agents and toxins 1. Reversible: EI complex can dissociate rapidly. Different kinds: can be distinguished kinetically 2. Irreversible: inhibitor becomes tightly bound to enzyme - covalently or non-covalently EI complex dissociates very s l o w l y, if at all 12-12Have 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 inhibition 12-13 2. Uncompetitive: inhibitor binds to separate site on enzyme, but only to ES complex 3. Noncompetitive (“pure”): inhibitor binds to separate site on E or ES complex 1. 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 by steady-state kinetics 12-14 “TRANSITION STATE” S ICompetitive inhibition 12-15Km Kmapp Kmapp Effect of a competitive inhibitor on Km and Vmax 12-16Effect of a competitive inhibitor on Km and Vmax 12-17Examples of competitive inhibitors - of hydroxymethylglutaryl coenzyme A reductase - block cholesterol synthesis S-CoA O 12-18Uncompetitive inhibition 12-19Effect of uncompetitive inhibitor on Km and Vmax Km and Vmax reduced by equivalent amounts In UNcompetitive inhibition, I binding pulls equilibrium towards ESI and therefore favors ES formation, i.e. as ES disappears, more E + S associate to restore the equilibrium amount of ES. Increased tendency of S to associate with E means Km ⇓. 12-2012-21 Noncompetitive inhibition ORIn NON-competitive inhibition, ki and kiʼare equal and cancel out one anotherʼs effect on Km, although Vmax goes down because active E molecules are being removed from the reaction mix. Effect of noncompetitive inhibitor on Km and Vmax 12-22The 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 0 0.222 0.0067 0 0.242 0.01 0 0.274 0.02 0 --------------------------------------------------------------------------------------------- V0 [S] [I] (M min-1) (M) (M) 0.162 0.005 0.01 0.185 0.0067 0.01 0.216 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 inhibition 12-23Solving 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
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