title: Proline iminopeptidase from the outer cell envelope of the human oral spirochete Treponema denticola ATCC 35405.
abstract: Certain periodontopathic organisms have been shown to exhibit high activity of proline iminopeptidase (PIPase). The human oral spirochete Treponema denticola ATCC 35405 was found to contain an easily extractable, 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 monomeric 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 cleaves specifically the Pro-Y bond in dipeptides where Y is preferably Arg or Lys. Pro-Gln, Pro-Asn, and Pro-Ala were also good substrates, while Pro-Glu was hydrolyzed slowly and Pro-Asp was not hydrolyzed at all. Tripeptides were poor substrates or were not hydrolyzed (an exception 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 PIPase may depend on an active carboxyl group and on an active seryl residue but not on metal cations. Diethylpyrocarbonate inactivated the enzyme in a reaction that was not reversible upon addition of NH2OH. The enzyme contains a relatively large percentage (ca. 15%) of proline residues. The dominance of the PIPase activity among aminopeptidase activities present in T. denticola and the proposed location of the enzyme in the outer cell envelope suggest that it has a vital function in the propagation of the cells within their biological niche (inflamed human periodontal tissues). The biologic role of the PIPase may be envisaged as in the termination of the overall peptidolytic cascade (liberating free proline and other amino acids), whereby host tissue proteins and peptides are first processed and inactivated by other peptidases possibly present within the same confines as the PIPase.
authors.affiliation: Department of Biologic and Materials Sciences, School of Dentistry, The University of Michigan, Ann Arbor, 48109-1078, USA.
authors.forename: K K
authors.initials: KK
authors.lastname: Mäkinen
authors.forename: C Y
authors.initials: CY
authors.lastname: Chen
authors.forename: P L
authors.initials: PL
authors.lastname: Mäkinen
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009055:Mouth / Q000382:microbiology*; D013379:Substrate Specificity; D013696:Temperature; D014208:Treponema / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D000626:Aminopeptidases; C022450:prolyl aminopeptidase