The Effect of the Leaving Group in the Acylation of α-Chymotrypsin byN-Acylimidazoles: The Reaction ofN-(3,3-Dimethylbutyryl)-4(5)-nitroimidazole

The kinetic and mechanistic effects of substitution in the leaving group ofN-acylimidazoles in α-chymotrypsin-catalyzed hydrolysis reactions have been investigated. The values ofk2/Kmhave been determined for acylation of the enzyme by a series ofN-(3,3-dimethylbutyryl)-4(5)-substituted imidazoles at 30°C. The plot of logk2/Kmvs pH for acylation byN-(3,3-dimethylbutyryl)-4(5)-nitroimidazole has regions of slope 1.0 and 0 with a pKappof 6.5, in contrast with the nearly pH-independent profiles forN-acylimidazoles without electron-withdrawing substituents in the leaving group. The effect of electron withdrawal is to lower the pKaof the conjugate acid of theN-acylimidazole, which thereby restricts the acylation reaction of that species. The pH independence of the acylation reactions ofN-acylimidazoles without electron withdrawal in the leaving group must reflect reactions of the neutral and protonated species that proceed with similar rate constants. The pKappof 6.5 is constant through wide structural variations and is therefore not due to the kinetics of the reaction, but is very likely the pKaof the conjugate acid of His-57. There is only a small effect of the pKaof the leaving group in the acylation reactions with β1g= −0.3. Therefore, there can only be moderate C–N bond breaking in the critical transition state. A plot of logk2/Kmvs σm, the Hammett substituent constant formetasubstituents, is linear with a slope, ρ, of 2.6 (r= 0.98). Whenparasubstituent constants are employed the ρ value is 2.1. The effect of substitution in the leaving group is nearly identical in the enzymatic acylation reaction and in the OH−-catalyzed hydrolysis of these compounds in regard to the values of ρ and β1g. Thus, the transition states in the two reactions must be similar in regard to the extent of C–N bond breaking and C–O bond formation with the nucleophile. The transition state very likely resembles the reactants.