Subsite preferences of pepstatin-insensitive carboxyl proteinases from bacteria

Pseudomonas sp. 101 carboxyl proteinase (PCP) and Xanthomonas sp. T-22 carb oxyl proteinase (XCP), the first and second unique carboxyl proteinases fro m prokaryotes to be isolated and characterized, are not inhibited by the cl assical carboxyl proteinase inhibitor pepstatin. In this study, we elucidat ed their subsite preferences by using a series of synthetic chromogenic sub strates, Lys-Pro-Ile(P-3)-Glu(P-2)-Phe*Nph-Arg(P-2')-Leu (P-3') (Nph is p-n itrophenylalanine, Phe*Nph is the cleavage site) with systematic substituti ons 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=6.0 5 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 sug gested that the S-2' subsites of PCP and XCP are occupied by hydrophilic re sidues, similar to that of pepstatin-insensitive carboxyl proteinase from B acillus coagulans J-4 [Shibata et al. (1998) J. Biochem. 124, 642-647]. In contrast, the S-2' subsite of pepstatin-sensitive carboxyl proteinases (asp artic proteinases) is hydrophobic in nature. Thus, the hydophilic nature of the S-2' subsite appears to be a distinguishing feature of pepstatin-insen sitive carboxyl proteinases.