ACYL TRANSFER-REACTIONS CATALYZED NATIVE AND MODIFIED ALPHA-CHYMOTRYPSIN IN ACETONITRILE WITH LOW WATER-CONTENT

The characterization of the S'subsite specificity of native and ethyla ted alpha-chymotrypsin has been studied via acyl transfer reaction in acetonitrile containing 10 vol% of water. Using Ac-Tyr-OEt as acyl don or, we investigated the partitioning of acyl-chymotrypsins between wat er and amino acid and peptide-derived nucleophiles. For the investigat ion of S'(2) subsite specificity, a series of 19 dipeptides of the gen eral structure Ala-Xaa (Xaa represents all natural amino acids except cysteine) were used. From the values of the apparent partition constan ts P-app, the order of preference for the P'(2) position is estimated to be: positively charged > hydrophilic greater than or equal to hydro phobic > aromatic > Pro > negatively charged side chain. Concerning th e S'(1) specificity, the same preference is deduced based on the study with the series of amino acid amides and Xaa-Ala dipeptides. In contr ast to the nucleophilic specificity of alpha-chymotrypsin in aqueous s olutions, free dipeptides and hydrophilic amino acid derivatives as nu cleophiles exhibit much higher reactivities for acyl transfer in aceto nitrile. We have not observed a significant difference in nucleophilic specificity between native and ethylated chymotrypsin.