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, a-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. alpha-Lytic protease and cercarial protease exhibit very little specificity toward the P'3 side chain, but the efficiency of nucleophiles with D-Ala or Gly in P'3 position was significantly reduced compared to that of nucleophiles with other P'3 side chains. These results suggest significant enzyme-nucleophile contacts beyond the S'3 subsite. Acyl transfer to alanine peptides of varying length revealed the existence of four S' subsites for alpha-lytic protease and at least five S' subsites for cercarial protease.