Role of the S' subsites in serine protease catalysis. Active-site mapping of rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni.
The S' subsite specificity of four homologous serine proteases, rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni, was studied by measuring acyl-transfer reactions to 100 pentapeptide nucleophiles. Peptides of the general structures H-Xaa-Ala-Ala-Ala-Ala-NH2, H-Ala-Xaa-Ala-Ala-Ala-NH2, and H-Ala-Ala-Xaa-Ala-Ala-NH2 were synthesized, where Xaa is D-Ala, Cit, and all natural amino acids except Cys. The variable residues of these nucleophiles occupy the P'1, P'2, and P'3 positions in acyl-transfer reactions. The P'1 and P'2 residues were found to influence the efficiency of the nucleophiles by more than 2 orders of magnitude, whereas the S'3 subsite shows a lower specificity in all four enzymes. We synthesized consensus peptides of the general structure H-aa1-aa2-aa3-Ala-Ala-NH2, in which two or three positions were occupied by amino acids that showed the highest specificity in the first series of nucleophiles. Peptides with optimal amino acid residues in the P'2 and P'3 positions show a very high efficiency in chymotrypsin- and trypsin-catalyzed reactions. Otherwise, large specific side chains in the P'1 and P'3 positions of the nucleophiles show less than additive binding contributions due to steric hindrance. Comparison of chymotrypsin-catalyzed acyl-transfer reactions to nucleophiles of the structures H-Xaa-Leu-Arg-Ala-Ala-NH2 and H-Xaa-Ala-Ala-Ala-Ala-NH2 reveals a significantly different P'1 specificity for both series which confirms steric hindrance between large P'1 and P'3 residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Schellenberger
V
V
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Turck
C W
CW
Rutter
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D015801:Helminth Proteins; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D051381:Rats; D012550:Schistosoma mansoni / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D015801:Helminth Proteins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C021826:myxobacter alpha-lytic proteinase; D014357:Trypsin; D003546:Cysteine Endopeptidases