Subsite preferences of pepstatin-insensitive carboxyl proteinases from bacteria

Pseudomonas sp. 101 carboxyl proteinase (PCP) and Xanthomonas sp. T-22 carboxyl proteinase (XCP), the first and second unique carboxyl proteinases from prokaryotes to be isolated and characterized, are not inhibited by the classical carboxyl proteinase inhibitor pepstatin. In this study, we elucidated their subsite preferences by using a series of synthetic chromogenic substrates, Lys-Pro-Ile(P-3)-Glu(P-2)-Phe*Nph-Arg(P-2')-Leu (P-3') (Nph is p-nitrophenylalanine, Phe*Nph is the cleavage site) with systematic substitutions at the P-3, P-2, P-2', and P-3' positions. Among 45 substrates tested, the best substrate for PCP had a Leu replacement at the P-2 position (k(cat) =27.2 s(-1), K-m=4.22 mu M, k(cat)/K-m =6.43 mu M-1 s(-1)), and that for XCP had an Ala replacement at the P-3 position (k(cat) =79.4 s(-1), K-m=6.05 mu M, k(cat)/K-m =13.1 mu M-1 s(-1)). PCP and XCP preferred such charged amino acid residues as Glu, Asp, Arg, or Lys at the P-2' position. This suggested that the S-2' subsites of PCP and XCP are occupied by hydrophilic residues, similar to that of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [Shibata et al. (1998) J. Biochem. 124, 642-647]. In contrast, the S-2' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydophilic nature of the S-2' subsite appears to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases.