Subsite preferences of pepstatin-intensitive carboxyl proteinases from prokaryotes: Kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase

Kumamolysin, a carboxyl proteinase from Bacillus novosp, MN-32, is characte rized by its thermostability and insensitivity to aspartic proteinase inhib itors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epo xy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidat ed using two series of synthetic chromogenic substrates: P-5-P-4-P-3-P-2-Ph e*Nph (p-nitrophenylalanine: *cleavage site)-P-2'-P-3', in which the amino acid residues at the P-5-P-2, P-2' and P-3' positions were systematically s ubstituted, Among 74 substrates, kumamolysin was shown. to hydrolyze Lys-Pr o-ne-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this p eptide were K-m = 41 +/- 5 mu M, k(cat) = 176 +/- 10 s(-1), and k(cat)/K-m = 4.3 +/- 0.6 mu M-1.s(-1). These systematic analyses revealed the followin g features: (i) Kumamolysin had a unique preference for the P-2 position. K umamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P-2 position; this was also observed for the pepstatin-insensitive c arboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al, (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomona s sp, 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas s p, T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P-2 pos ition. (ii) Kumamolysin preferred such charged amino acid residues as Glu o r Arg at the P-2' position, suggesting that the S-2' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4, In general, the S-2' subsite of pepstatin-sensitive carboxyl proteinases ( aspartic proteinases) is hydrophobic in nature, Thus, the hydrophilic natur e of the S-2' subsite was confirmed to be a distinguishing feature of pepst atin-insensitive carboxyl proteinases from prokaryotes.