NUCLEOPHILE SPECIFICITY OF SUBTILISIN IN AN ORGANIC-SOLVENT WITH LOW WATER-CONTENT - INVESTIGATION VIA ACYL TRANSFER-REACTIONS

Nucleophile specificity of subtilisin (subtilopeptidase A) was studied via acyl transfer reactions in acetonitrile containing piperidine and 10 vol% of water. Ac-Tyr-OEt was used as acyl donor and a series of a mino acid derivatives, di- and tripeptides of the general structure Xa a-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) were used as nucleophiles. The nucleophilic efficienc ies of these peptides were characterized by the values of the apparent partition constants, rho(app), determined from the HPLC analysis of t he reactions. The order of preference for the P-1 position was estimat ed to be: Gly > hydrophilic, positively charged > hydrophobic, aromati c > negatively charged > Leu >>> Pro side chain. For the P-2 position the order of preference was: Gly > hydrophilic, charged > hydrophobic, aromatic > Pro side chain. The values of rho(app) for Gly-Gly-Xaa tri peptides cover a range of only two orders of magnitude, with lower nuc leophile efficiency for those with hydrophobic amino acid residues in the P-3 position. The dipeptide with Pro in P-1 did not react at all, but a tripeptide having Pro in P-3 was a very good nucleophile. The ne gatively charged amino acid residues in the P-1 position result in ver y weak nucleophilic behavior, whereas the peptides with Asp or Glu in P-2 and P-3 are well accepted. Generally, peptides of the Gly-Xaa or G ly-Gly-Xaa series were better nucleophiles than peptides of the Xaa-Gl y series. The length of the peptide chain or amidation of alpha-carbox yl function had no influence on nucleophilic behavior. No significant difference in nucleophile specificity between subtilopeptidase A and n agarse was observed. 1996 (C) John Wiley & Sons, Inc.