Kinetic study of the alpha-chymotrypsin-catalyzed hydrolysis and synthesis of a peptide bond in a monophasic aqueous/organic reaction medium.

We have studied the hydrolysis and synthesis reactions of the peptide bond involved in N-Cbz-L-tryptophanyl-glycineamide as catalyzed by alpha-chymotrypsin in various mixtures of water and 1,4-butanediol. Using a constant nonsaturating concentration of substrate, the initial reaction rates decreased exponentially with decreasing water content in the solvent mixture. When the water content was decreased from 100 to 20% (v/v), the maximum rate of reaction did not vary by more than a factor of 2 to 4, whereas the Michaelis constant increased exponentially. This exponential variation of the Michaelis constant was due to changes in the partitioning of the substrate between the active site of the enzyme and the solvent. In these processes, the actual rate constants did not vary when the relative contents of water and 1,4-butanediol were varied.