Purification and substrate specificity of an endopeptidase from the human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-Leu-Gly-Pro-Ala and bradykinin.
An endopeptidase was purified to homogeneity from the cell extracts of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised dialysis, anion exchange fast protein liquid chromatography (FPLC), hydroxylapatite FPLC, immobilized metal affinity FPLC, FPLC chromatofocusing, and two consecutive gel permeation FPLC steps. The enzyme is a 62-kDa protein with an isoelectric point of 6.5-7.0. Experiments with enzyme inhibitors suggest that this enzyme is a metallopeptidase and that its activity is not dependent on sulfhydryl or serine residues. The enzyme is active on furylacryloyl-Leu-Gly-Pro-Ala (FALGPA; pH optimum near 6.25), bradykinin (Bk), and several Bk-related peptides. In FALGPA, the cleavage site is the Leu-Gly bond. An imino acid is absolutely necessary in position P'2. The shortest hydrolyzed peptide was FALGPA, the hydrolysis of which is strongly and competitively inhibited by Bk (Ki = 5.0 microM). The pyrophosphate ion and phosphoramidon also inhibited the hydrolysis of FALGPA. The enzyme does not hydrolyze all typical synthetic collagenase substrates, Azocoll, Azocasein, or Type I and Type IV collagens, or any other proteins tested. In Bk-related peptides, the hydrolyzed bond was Phe5-Ser6. Since a Bk antagonist and a Bk-potentiating pentapeptide also were good substrates, it is possible that the enzyme hydrolyzes Bks and related peptides only because of the coincidental, specific amino acid sequence of those substrates. A proposal is made that since a substantial portion of the amino acid sequence of FALGPA is present in collagen (and additionally acknowledging that the furylacryloyl residue structurally resembles that of proline), the natural substrates of this enzyme may be small, soluble collagen fragments produced by other enzymes from periodontal connective tissue, and that such peptides are important for the nutrition and pathogenicity of T. denticola.
Mäkinen
K K
KK
Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor 48109-1078.
Mäkinen
P L
PL
Syed
S A
SA
D000179:Acrylates / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001920:Bradykinin / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D017886:Durapatite; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010514:Periodontal Pocket / Q000382:microbiology; D013379:Substrate Specificity; D014208:Treponema / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification
D000179:Acrylates; D000596:Amino Acids; D006882:Hydroxyapatites; D009842:Oligopeptides; C031821:2-furanacryloyl-leucyl-glycyl-prolyl-alanine; D017886:Durapatite; C103267:FALGPA peptidase; D008666:Metalloendopeptidases; D001920:Bradykinin