THE 2ND NUCLEOPHILE MOLECULE BINDS TO THE ACYL-ENZYME-NUCLEOPHILE COMPLEX IN ALPHA-CHYMOTRYPSIN CATALYSIS - KINETIC EVIDENCE FOR THE INTERACTION

Alpha-Chymotrypsin-catalyzed acyl tranfer 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.