Mapping the S′ Subsites of Serine Proteases Using Acyl Transfer to Mixtures of Peptide Nucleophiles

Abstract

We have developed a rapid and convenient procedure for the characterization of the S′ subsite specificity of serine proteases. A mixture of peptide nucleophiles is incubated with the enzyme in the presence of excess of a specific ester substrate. The decrease in each nucleophile concentration is monitored by high-performance liquid chromatography analysis of the dansylated mixture. Relative kinetic parameters for each nucleophile in the mixture are then calculated using a new statistical algorithm that relates all pairs of nucleophiles. As a first application, we investigated the S′1 subsite specificity of chymotrypsin, trypsin, and a recently described trypsin mutant, Tr → Ch[S1 + L1 + L2] with chymotrypsin-like primary specificity [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255,1249-1253], For this purpose 21 peptide nucleophiles of the general structure H-Xaa-Ala-Ala-Ala-Ala-NH2 were prepared by multiple solid-phase synthesis, where Xaa represents d-alanine, citrulline, and all natural amino acids except cysteine. Relative second-order rate constants for the enzyme-catalyzed acyl transfer to these nucleophiles were determined over a range of 102. Chymotrypsin and trypsin have markedly different S′1 specificities. The order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic > aromatic ≫ negatively charged, d-Ala, Pro P′1 side chain. Trypsin prefers hydrophobic residues, but like chymotrypsin aliphatic residues are better than aromatic residues in P′1 position. The S′1 specificity of the mutant Tr → Ch[S1 + L1 + L2] is similar to the specificity of trypsin; however, P′1 aromatic residues have low reactivity characteristic of chymotrypsin. © 1993, American Chemical Society. All rights reserved.