Characterization of the S′-subsite specificity of bovine pancreatic α-chymotrypsin via acyl transfer to added nucleophiles.

The S′-subsite specificity of bovine pancreatic α-chymotrypsin was investigated by acyl transfer reactions using a series of amino-acid- and peptide-derived nucleophiles. The nucleophilic efficiency covers a range of more than three orders of magnitude, reflecting the specificity of the acyl transfer process. Positively charged H-Arg-NH₂ was the most efficient nucleophile of the series while peptides with free carboxyl groups show poor nucleophilic behaviour. This is explained by electrostatic interactions with the residues Asp35 and Asp64 of the enzyme. These negatively charged groups, which are localized near the appropriate S′ binding sites, repel carboxylate groups of the nucleophiles. There is a good correlation between the nucleophile efficiencies found for different acyl enzymes. An investigation of a series of 14 water-soluble acyl donor esters, differing both in the P₁ residue and in the number of amino acids, revealed that the nature of the acyl group affected the acyl-enzyme partitioning between water and added nucleophile in the range of one order of magnitude. [ABSTRACT FROM AUTHOR]