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 s eries of amino-acid amides. Most of the reactions studied can be descr ibed 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 line ar dependence of the partition constant, p, on the nucleophile concent ration which can be interpreted by the hydrolysis of the acyl-enzyme-n ucleophile complex. The alpha-chymotrypsin-catalyzed transfer of the B Z-L-Tyr and Ac-L-Trp groups to several amino-acid amides showed unusua l behavior which can be interpreted by the kinetic model involving for mation 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.