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'i subsite specificity of chymotrypsin,
trypsin, and a recently described trypsin mutant, Tr -*· Ch[Sl + LI + 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'i specificities. The
order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic >
aromatic » negatively charged, o-Ala, Pro P'i side chain. Trypsin prefers hydrophobic residues, but like
chymotrypsin aliphatic residues are better than aromatic residues in P'i position. The S'i specificity of the
mutant Tr — Ch[Sl + LI + L2] is similar to the specificity of trypsin; however, P'i aromatic residues have
low reactivity characteristic of chymotrypsin.