Kinetics of a Thermolysin-Catalyzed Peptide Formation Reaction in Acetonitrile–Water

Thermolysin-catalyzed peptide condensation reactions from acylamino acids and amino acid amides were studied in an acetonitrile (MeCN)–water mixed solvent in a homogeneous system. A kinetic analysis of the condensation reaction was performed by taking into account the simultaneous, solvent-induced, gradual inactivation of the enzyme. Although the dependence on the concentration of the carboxyl component gave linear double-reciprocal plots, that on the amine component at 70% MeCN showed an apparent substrate inhibition profile. An analysis of the apparent Km and kcat parameters for both components at 40% MeCN indicated that the condensation reaction proceeded by a random Bi–Bi mechanism. The Km values were estimated to be 5.5 mM for Cbz–Phe and 110 mM for LeuNH2, and kcat was 4—5 s−1. The Km values for Cbz–Phe and LeuNH2 were also evaluated at 70% MeCN to be 5 and 100—150 mM, respectively. This indicates that the catalytic property of this enzyme is not significantly influenced by the existence of a higher concentration of such an organic solvent, except for the second nonproductive binding of the amine component.