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-b y a procedure that comprised a mild Triton X-100 extraction (which dis integrates the outer membrane but leaves the cells morphologically int act) and four successive fast protein liquid chromatographic steps of the extract. The activity of this oligopeptidase (formerly named ''try psin-like'' enzyme and ''BANA-peptidase'') together with the proteinas e 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-associate d 78-kDa protein with an isoelectric point of 6.1, and its estimated m inimum peptide length was 688 amino acid residues. The OPase does not hydrolyze proteins, but hydrolyzes -X-Arg-p-nitroaniline peptides betw een 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 subs trates. Ca(II) did not affect the values of the Michaelis constant. Th e OPase activity is not dependent on reactive SH-groups, but is sugges ted to depend on he catalytic triad COOH...His...Ser. The N-terminal s equence for the first 29 amino acid residues is MKQSDFEKPPIAEIKETRFEKF GKTRIDN. The purified enzyme is very sensitive to chlorhexidine acetat e (mixed inhibition; K-i = 0.85 mu M) and somewhat less sensitive to b acitracin (K-i(app) = 27.5 mu M). The present OPase is considered to b elong 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 rese mbles to a certain extent that of some members of the coagulation casc ade. (C) 1995 Academic Press, Inc.