The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis

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Abstract
α-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 α-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 α-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 α-chymotrypsin-catalyzed acyl transfer evident in previous studies.

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