Acyl transfer reactions catalyzed by native and modified alpha-chymotrypsin in acetonitrile with low water content.

The characterization of the S' subsite specificity of native and ethylated alpha-chymotrypsin has been studied via acyl transfer reaction in acetonitrile containing 10 vol% of water. Using Ac-Tyr-OEt as acyl donor, we investigated the partitioning of acyl-chymotrypsins between water and amino acid and peptide-derived nucleophiles. For the investigation of S'2 subsite specificity, a series of 19 dipeptides of the general structure Ala-Xaa (Xaa represents all natural amino acids except cysteine) were used. From the values of the apparent partition constants rho app, the order of preference for the P'2 position is estimated to be: positively charged > hydrophilic > or = hydrophobic > aromatic > Pro > negatively charged side chain. Concerning the S'1 specificity, the same preference is deduced based on the study with the series of amino acid amides and Xaa-Ala dipeptides. In contrast to the nucleophilic specificity of alpha-chymotrypsin in aqueous solutions, free dipeptides and hydrophilic amino acid derivatives as nucleophiles exhibit much higher reactivities for acyl transfer in acetonitrile. We have not observed a significant difference in nucleophilic specificity between native and ethylated chymotrypsin.