Effects of Normal and Their Branched Alcohols with Structurally Minimal Variation on Kinetic Parameters in Thermolysin-catalyzed Peptide Hydrolysis and Synthesis of N-(Benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine and Its Methyl Ester

Abstract

When higher alcoholic solvents were added to the reaction medium, and the enhancement of the enzyme activity, followed by its reduction then inactivation, were observed in thermolysin-catalyzed peptide hydrolysis and synthesis. The organic solvent content used was less than the saturating concentration in the buffer (i.e., water-organic one-phase system). The kinetic parameters, K_m and k_cat, at the alcoholic concentration giving maximal enzyme activity in these reactions changed linearly with increasing log P values of the alcohols and consequently k_cat/K_m as well. When the branched isomers of alcohols with structurally minimal variation of which log P was equivalent theoretically, were used as annexments, the kinetic parameters were also changed. The results, especially the changes of K_m for each organic solvent, suggested that each alcohol should act at the active site of the enzyme in its own effective mode.