Identification of Carboxyl Residues in Pepstatin-Insensitive Carboxyl Proteinase from Pseudomonas sp. 101 that Participate in Catalysis and Substrate Binding

Abstract

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, is the first example from a prokaryote of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1, 2-epoxy-3(p-nitrophenoxy)propane. To identify the catalytic residue(s) of PCP, chemical modification was carried out using carboxyl residue-specific reagents, carbodiimides. PCP was inactivated effectively by N, N'-dicyclohexylcarbodiimide (DCCD) with pseudo-first-order kinetics. For the inactivation, 0.7 mol DCCD was involved per 1 mol PCP. The effects of pH and methanol on the inactivation showed that two carboxyl residues (Asp and/or Glu) were involved in the reaction. The inactivation by DCCD was prevented by a competitive inhibitor, tyrostatin, or a synthetic substrate in a concentration-dependent manner. Based on these data, differential labeling of PCP with DCCD was carried out: Firstly, PCP was treated with cold DCCD in the presence of tyrostatin. After removal of the tyrostatin, which covered the substrate binding site, by dialysis, the PCP was treated with [¹⁴C]DCCD to label carboxyl residue(s) essential for its function. Two labeled peptides were isolated by HPLC from a trypsin digest of cold- and [¹⁴C]DCCD modified PCP. On analysis of their amino acid sequences, it was revealed that the [¹⁴C]-DCCD was bound to Asp¹⁴⁰ and Glu²²² of PCP, respectively. Based on these data, it was strongly suggested that Asp¹⁴⁰ and Glu²²² of PCP were involved in its catalytic function or substrate binding.