Identification of carboxyl residues in pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 that participate in catalysis and substrate binding.
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.
Ito
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Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto, 606-8585, Japan.
Narutaki
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Uchida
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Oda
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D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D020134:Catalytic Domain; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010436:Pepstatins / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009842:Oligopeptides; D010436:Pepstatins; D010455:Peptides; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; C074130:tyrostatin; D001224:Aspartic Acid; D018698:Glutamic Acid; D004024:Dicyclohexylcarbodiimide; D012697:Serine Endopeptidases; C104058:xanthomonapepsin; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin; D000432:Methanol