Mapping the S' subsites of serine proteases using acyl transfer to mixtures of peptide nucleophiles.
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 10(2). 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.
Schellenberger
V
V
Hormone Research Institute, University of California, San Francisco 94143.
Turck
C W
CW
Hedstrom
L
L
Rutter
W J
WJ
D000217:Acyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009842:Oligopeptides; D011994:Recombinant Proteins; D000217:Acyltransferases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin