The second nucleophile molecule binds to the acyl-enzyme-nucleophile complex in alpha-chymotrypsin catalysis. Kinetic evidence for the interaction.

alpha-Chymotrypsin-catalyzed acyl transfer was studied using three acyl-group donors (Mal-L-Ala-L-Ala-L-PheOMe, Bz-L-TyrOEt and Ac-L-TrpOEt; Mal, maleyl; Bz, benzoyl; OMe, methyl ester; OEt, ethyl ester) and a series of amino-acid amides. Most of the reactions studied can be described by the simplest kinetic model without the nucleophile binding to the acyl-enzyme. The alpha-chymotrypsin-catalyzed transfer of the Mal-L-Ala-L-Ala-L-Phe group to the amides of L-Phe and L-Tyr showed a linear dependence of the partition constant, p, on the nucleophile concentration which can be interpreted by the hydrolysis of the acyl-enzyme-nucleophile complex. The alpha-chymotrypsin-catalyzed transfer of the Bz-L-Tyr and Ac-L-Trp groups to several amino-acid amides showed unusual behavior which can be interpreted by the kinetic model involving formation of a complex of the acyl-enzyme with two nucleophile molecules. These observations can explain the conflicting conclusions concerning the kinetics of alpha-chymotrypsin-catalyzed acyl transfer evident in previous studies.