EFFECTS OF SUBZERO TEMPERATURES ON THE KINETICS OF PROTEASE-CATALYZEDDIPEPTIDE SYNTHESIS IN ORGANIC MEDIA

A dipeptide synthesis was drastically influenced by the reaction tempe rature, in the range from -30 degrees to 25 degrees C. This article sh ows the kinetic reasons of this effect. alpha-Chymotrypsin was immobil ized on celite and used in four different water-miscible solvents cont aining small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamid e (Ala-NH2). In the competition between the nucleophile (Ala-NH2) and water for the acylenzyme complex, the nucleophile was favored by low r eaction temperatures. This effect (quantified as p-values) was observe d in all four solvents, and it was greatest in acetonitrile and tetrah ydrofuran. The esterase and amidase activities of the enzyme were stud ied using Ac-PheOEt and N-acetyl-L-phenylalanyl-L-alaninamide (Ac-PheA la-NH2) as substrates. The Michaelis-Menten parameters, K-m,K-app and V-max, were determined for ester hydrolysis and dipeptide hydrolysis. Both K-m,K-app and V-max tended to increase with increasing temperatur e. Secondary hydrolysis was reduced at subzero temperatures because es ter hydrolysis was favored in relation to dipeptide hydrolysis. Dipept ide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enz yme; and, second, in the competition between the ester substrate and t he peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20 degrees C comp ared with 84% at 25 degrees C. (C) 1995 John Wiley & Sons, Inc.