PROLINE IMINOPEPTIDASE FORM THE OUTER CELL-ENVELOPE OF THE HUMAN ORALSPIROCHETE TREPONEMA-DENTICOLA ATCC-35405

Certain periodontopathic organisms have been shown to exhibit high act ivity of proline iminopeptidase (PIPase). The human oral spirochete Tr eponema denticola ATCC 35405 was found to contain an easily extractabl e, novel PIPase (EC 3.4.11.5), which was purified to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis-pure form by means of fast protein liquid chromatographic procedures. The range of the minimum mo nomeric molecular mass (280 amino acid residues) of the PIPase, based on amino acid analysis, was 30.35 to 30.39 kDa, but the likely in vivo form of the enzyme is a tetramer (minimum mass, 120.2 to 120.4 kDa). The molecular masses based on laser desorption mass spectrometry were 36.058 kDa for the monomer and 72.596 kDa for a dimer. The PIPase clea ves specifically the Pro-Y bond in dipeptides where Y is preferably Ar g or Lys. Pro-Gln, Pro-Asn, and Pro-Ala were also good substrates, whi le Pro-Glu was hydrolyzed slowly and Pro-Asp was not hydrolyzed at all . Tripeptides were poor substrates or were not hydrolyzed (an exceptio n was Pro-Gly-Gly, which cleaved at a moderate rate). Larger molecules , such as poly-L-Pro, were not hydrolyzed. The T. denticola enzyme can be regarded as a true PIPase, since replacing Pro in Pro-Y with other amino acid residues resulted in no hydrolysis. The activity of the PI Pase may depend on an active carboxyl group and on an active seryl res idue but not on metal cations. Diethylpyrocarbonate inactivated the en zyme in a reaction that was not reversible upon addition of NH2OH. The enzyme contains a relatively large percentage (ca. 15%) of proline re sidues. The dominance of the PIPase activity among aminopeptidase acti vities present in T. denticola and the proposed location of the enzyme in the outer cell envelope suggest that it has a vital function in th e propagation of the cells within their biological niche (inflamed hum an periodontal tissues). The biologic role of the PIPase may be envisa ged as in the termination of the overall peptidolytic cascade (liberat ing free proline and other amino acids), whereby host tissue proteins and peptides are first processed and inactivated by other peptidases p ossibly present within the same confines as the PIPase.