Substituent Effects on Substrate Activation and Michaelis-Menten Kinetic Paramenters in the α-Chymotrypsin-catalyzed Hydrolysis of Phenyl Acetates

Abstract

The effects of substituents on the steady state and pre-steady state kinetics in α-chymotrypsin [EC 3. 4. 21. l]-catalyzed hydrolysis were studied using substituted phenyl acetates. In the steady state hydrolysis, substrate activation, which had been observed and studied previously for p-nitrophenyl acetate, was also observed for p-broma-, p-chloro-, and m-methylphenyl acetates. Little activation was observed for p-acetyl-, m-nitro-, p-methyl-, and p-methoxyphenyl acetates. Addition of p-dichlorobenzene increased k_cat for all substrates examined and greatly diminished the substrate activation for the activatable substrates. This suggests that substrate activation is brought about by binding of the substrate(s) to activator binding site(s).
The value of k_cat decreased in accordance with increase of the σ^- value of substituents. On the other hand, k_cat/K_m(app) showed an opposite σ^- dependence, as was previously observed. In pre-steady state measurements, little burst was observed for more electron-donating substituents than m-nitro. The σ^- dependence of k_cat is apparently not consistent with the prediction derived from that of k_Cat/K_m(app) on the basis of the usual two-step mechanism with a common acetyl-enzyme intermediate.