12 1 MCB 450 Lecture 12 Catalytic strategies used by enzymes Inhibition of enzyme activity Example of an enzyme mechanism peptide hydrolysis by chymotrypsin 12 2 Catalytic strategies used by enzymes 1 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 energy 4 Catalysis by approximation orientation Enzyme brings two substrates close together and orients the reacting parts of substrate molecules for reaction 12 3 Covalent catalysis Catalysis through formation of covalent bonds between E and S Substrate nucleophilic attack by a group on E on a carbonyl carbon Nucleophiles Functional groups rich in ethat can combine with and give up e to e deficient electrophiles E is covalently linked to the substrate acyl enzyme intermediate 12 4 General acid base catalysis 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 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 12 5 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 6 Zinc in the metalloprotease thermolysin Water molecule activated to serve as nucleophile attacks carbonyl carbon of peptide bond 12 7 Catalysis by approximation orientation Enzyme E Bond 12 8 Effect of heat on enzyme activity SS LO N E TI O U R RA CT TU RU N A ST DE 3 D OF LY N E IO LIK OT E M R AL MO RM NS E O TH CTI ES R A S E EA I N T R C I N E S AT ING E H AK M 12 9 Effect of pH on enzyme activity Ability of an enzyme to use the side chains of 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 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 2 is deprotonated Activity then goes down as critical higher pKa group on 1 is deprotonated 12 11 TWO CRITICAL IN ACTIVE SITE Interpretation 12 12 Inhibition 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 13 Types of reversible inhibition 1 Competitive inhibitor binds to enzyme s active site 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 Have 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 12 14 Competitive inhibitors TRANSITION STATE S Often resemble substrate structurally Compete with substrate for active site I 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 15 Competitive inhibition 12 16 Effect of a competitive inhibitor on Km and Vmax Km Kmapp Kmapp 12 17 Effect of a competitive inhibitor on Km and Vmax 12 18 Examples of competitive inhibitors of hydroxymethylglutaryl coenzyme A reductase block cholesterol synthesis S CoA O 12 19 Uncompetitive inhibition 12 20 Effect 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 21 Noncompetitive inhibition OR 12 22 Effect of noncompetitive inhibitor on Km and Vmax In NON competitive inhibition ki and ki are equal and cancel out one another s effect on Km although Vmax goes down because active E molecules are being removed from the reaction mix 12 23 Solving problems on enzyme inhibition The 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 12 24 Solving 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 Kmapp 12 25 REACTIVE AMINO ACID IN ACTIVE SITE IS COVALENTLY MODIFIED BY A GROUP SPECIFIC REAGENT NOT OTHER SERINES IN THE PROTEIN 12 26 12 27 DIPF acetylcholinesterase inhibitor DIPF Blocks normal deactivation of neurotransmitter acetylcholine Acetycholine accumulates nerve impulses can t be stopped get prolonged muscle contraction paralysis of respiratory muscles and death 12 28 Mechanism of enzyme catalysis chymotrypsin Cleaves peptide bonds C terminal to aromatic Enhances rate of peptide hydrolysis by 109 Reaction i illustrates transition state stabilization ii example of acid base catalysis and covalent catalysis Chymotrypsin i consists of 3 S S linked polypeptide chains total MW 25 000 ii 3D structure known Active site His57 Asp102 and Ser195 separated in primary sequence but grouped together in 3D structure 12 29 2 phases of the chymotrypsin reaction 1 Acylation Ser195 R OH oxygen acts as nucleophile peptide bond cleaved ester linkage formed between peptide carbonyl the enzyme 2
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