Purification and general properties of an oligopeptidase from Treponema denticola ATCC 35405--a human oral spirochete.

An endo-acting oligopeptidase (OPase) was purified to homogeneity from the cells of Treponema denticola ATCC 35405--a human oral spirochete--by a procedure that comprised a mild Triton X-100 extraction (which disintegrates the outer membrane but leaves the cells morphologically intact) and four successive fast protein liquid chromatographic steps of the extract. The activity of this oligopeptidase (formerly named "trypsin-like" enzyme and "BANA-peptidase") together with the proteinase activities of T. denticola and Porphyromonas gingivalis is utilized in a diagnostic test for human periodontal infections, but the enzyme's chemical nature has not been studied. The enzyme is a cell-associated 78-kDa protein with an isoelectric point of 6.1, and its estimated minimum peptide length was 688 amino acid residues. The OPase does not hydrolyze proteins, but hydrolyzes -X-Arg-p-nitroaniline peptides between arginine and the chromogen, the optimum pH of hydrolysis covering a broad pH range (7 to 9). The OPase is not a metalloenzyme, although 1.0 mmol/liter Ca(II) increases the rate of the hydrolysis of all substrates. Ca(II) did not affect the values of the Michaelis constant. The OPase activity is not dependent on reactive SH-groups, but is suggested to depend on the catalytic triad COOH. . .His. . .Ser. The N-terminal sequence for the first 29 amino acid residues is MKQSDFEKPPIAEIKETRFEKFGKTRIDN. The purified enzyme is very sensitive to chlorhexidine acetate (mixed inhibition; Ki = 0.85 microM) and somewhat less sensitive to bacitracin (Ki(app) = 27.5 microM). The present OPase is considered to belong to the serine peptidases, functionally resembling trypsin except that the OPase does not hydrolyze proteins. The OPase may be regarded as an oligopeptidase, the substrate specificity profile of which resembles to a certain extent that of some members of the coagulation cascade.