AN ENDO-ACTING PROLINE-SPECIFIC OLIGOPEPTIDASE FROM TREPONEMA-DENTICOLA ATCC-35405 - EVIDENCE OF HYDROLYSIS OF HUMAN BIOACTIVE PEPTIDES

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X- 100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 7 7-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydroly zed (optimum pH 65) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroan ilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in sev eral human bioactive peptides, such as bradykinin, substance P, neurot ensin, angiotensins, oxytocin, vasopressin, and human endothelin fragm ent 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2 P1P'(1), while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P-1 was absolutely necessary. The hydrolysis of Z -Gly-Pro-pNA was potently inhibited by the following, with the K-i(app ) (in micromolar) in parentheses: insulin B-chain (0.7), human endothe lin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and in hibition studies suggest that the POPase is a serine endopeptidase who se activity depends on the catalytic triad of COOH... Ser... His but n ot on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contai n a reactive cysteinyl residue near the active site. Amino acid residu es 4 to 24 of the first 24 N-terminal residues showed a homology of 71 % with the POPase precursor from Flavobacterium meningosepticum and co nsiderable homology with the Aeromonas hydrophila POPase. The ready hy drolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chroni city of periodontal infections by participating in the peptidolytic pr ocessing of those peptides.