S'-subsite mapping of polyethylene glycol-modified alpha-chymotrypsin and alpha-chymotrypsin: a comparative study.

Nucleophile specificities of polyethylene glycol-modified alpha-chymotrypsin and the native enzyme were investigated via acyl transfer reactions using Ac-Tyr-OEt as acyl donor and a large series of peptides and amino-acid amides as nucleophiles. In acyl transfer reactions with amino-acid amides both enzymes prefer basic and bulky amino-acid residues. However, peptides with bulky aliphatic or aromatic residues in P1' position were very poor nucleophiles for both enzymes. Surprisingly, peptides having bulky aliphatic or aromatic residues in P2' were preferred by the modified enzyme and were apparently more efficient nucleophiles for both enzymes than those with such residues in P1'. Generally, peptides with a longer chain were weaker nucleophiles in the reactions catalyzed by polyethylene glycol-modified enzyme. In the series of peptides containing a positively charged amino-acid residue in various locations, the order of nucleophilic efficiency is with this location being: P1' > P3' > P2'; this is valid for both enzymes.