Kinetic Study of α-Chymotrypsin Catalysis with Regard to the Interaction between the Specificity-determining Site and the Aromatic Side Chain of Substrates12

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Abstract
In order to investigate how changes in the structures of side-chain aromatic groups of specific substrates influence binding and kinetic specificity in α-chymotrypsin [EC 3.4.21. 1]-catalyzed reactions, a number of nucleus-substituted derivatives of the specific ester substrates were prepared and steady-state kinetic studies were carried out at pII 6.5 and 7.8. Ac-Trp(NCps)-OMe was hydrolyzed more readily at low substrate concentration than Ac-Trp-OMe due to its smaller Km(app) value, suggesting that the bulky 2-nitro-4-carboxyphenylsulfenyl moiety interacts with outer residues rather than with those in the hydrophobic pocket and that this interaction increases the binding specificity. Inhibition experiments using the corresponding carboxylate and analogous inhibitors, however, showed that the carboxy group at the para position of the phenyl nucleus of the substituent sterically hinders association with the active site of αchymotrypsin at pH 7.8 but not at pH 6.5. The kcat values of Ac-Trp(CHO)-OMe, Ac-Tyr(3-NO2) OMe, and Ac-m-Tyr-OMc were much higher than those of the corresponding specific substrates, indicating that derivatives with a substituent as large as a formyl, nitro or hydroxyl group at the ε-position are stereochemically favorable to the catalytic process. Remarkable increases in Km(app) were also observed. The individual parameters for Ac-Dopa-OMe, however, were comparable to those for Ac-Tyr-OMe.