Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 May 1;379(Pt 3):633–640. doi: 10.1042/BJ20031116

Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens.

Judit Marokházi 1, György Kóczán 1, Ferenc Hudecz 1, László Gráf 1, András Fodor 1, István Venekei 1
PMCID: PMC1224120  PMID: 14744262

Abstract

A proteolytic enzyme, Php-B ( Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens. The enzyme is intracellular, and its molecular mass is 74 kDa. Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6x10(2) s(-1), K(m)=5.8x10(-5) M(-1), pH optimum approx. 7.0). The p K(a1) and the p K(a2) values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion. The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P. luminescens by degrading small collagen fragments. For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis-Menten model to the experimental progress curves. Since this approach has not been used before in the characterization of proteases that are specific for the P1'-P4' substrate sites (e.g. collagenolytic enzymes), we present a comparison of this method with more conventional ones. The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.

Full Text

The Full Text of this article is available as a PDF (148.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barshop B. A., Wrenn R. F., Frieden C. Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system. Anal Biochem. 1983 Apr 1;130(1):134–145. doi: 10.1016/0003-2697(83)90660-7. [DOI] [PubMed] [Google Scholar]
  2. Blackburn M, Golubeva E, Bowen D, Ffrench-Constant RH. A novel insecticidal toxin from photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of manduca sexta . Appl Environ Microbiol. 1998 Aug;64(8):3036–3041. doi: 10.1128/aem.64.8.3036-3041.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bowen D., Blackburn M., Rocheleau T., Grutzmacher C., ffrench-Constant R. H. Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes. Insect Biochem Mol Biol. 2000 Jan;30(1):69–74. doi: 10.1016/s0965-1748(99)00098-3. [DOI] [PubMed] [Google Scholar]
  4. Bowen D., Rocheleau T. A., Blackburn M., Andreev O., Golubeva E., Bhartia R., ffrench-Constant R. H. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science. 1998 Jun 26;280(5372):2129–2132. doi: 10.1126/science.280.5372.2129. [DOI] [PubMed] [Google Scholar]
  5. Bowen DJ, Ensign JC. Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium photorhabdus luminescens . Appl Environ Microbiol. 1998 Aug;64(8):3029–3035. doi: 10.1128/aem.64.8.3029-3035.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Daborn P. J., Waterfield N., Blight M. A., Ffrench-Constant R. H. Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection. J Bacteriol. 2001 Oct;183(20):5834–5839. doi: 10.1128/JB.183.20.5834-5839.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dalhammar G., Steiner H. Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects. Eur J Biochem. 1984 Mar 1;139(2):247–252. doi: 10.1111/j.1432-1033.1984.tb08000.x. [DOI] [PubMed] [Google Scholar]
  8. Feder J. A spectrophotometric assay for neutral protease. Biochem Biophys Res Commun. 1968 Jul 26;32(2):326–332. doi: 10.1016/0006-291x(68)90389-6. [DOI] [PubMed] [Google Scholar]
  9. Fischer-Le Saux M., Viallard V., Brunel B., Normand P., Boemare N. E. Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov. Int J Syst Bacteriol. 1999 Oct;49(Pt 4):1645–1656. doi: 10.1099/00207713-49-4-1645. [DOI] [PubMed] [Google Scholar]
  10. Flyg C., Kenne K., Boman H. G. Insect pathogenic properties of Serratia marcescens: phage-resistant mutants with a decreased resistance to Cecropia immunity and a decreased virulence to Drosophila. J Gen Microbiol. 1980 Sep;120(1):173–181. doi: 10.1099/00221287-120-1-173. [DOI] [PubMed] [Google Scholar]
  11. Forst S., Dowds B., Boemare N., Stackebrandt E. Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu Rev Microbiol. 1997;51:47–72. doi: 10.1146/annurev.micro.51.1.47. [DOI] [PubMed] [Google Scholar]
  12. Gráf L., Jancsó A., Szilágyi L., Hegyi G., Pintér K., Náray-Szabó G., Hepp J., Medzihradszky K., Rutter W. J. Electrostatic complementarity within the substrate-binding pocket of trypsin. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4961–4965. doi: 10.1073/pnas.85.14.4961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guo L., Fatig R. O., 3rd, Orr G. L., Schafer B. W., Strickland J. A., Sukhapinda K., Woodsworth A. T., Petell J. K. Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins. Purification and characterization of toxin A and toxin B. J Biol Chem. 1999 Apr 2;274(14):9836–9842. doi: 10.1074/jbc.274.14.9836. [DOI] [PubMed] [Google Scholar]
  14. Harrington D. J. Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun. 1996 Jun;64(6):1885–1891. doi: 10.1128/iai.64.6.1885-1891.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hegyi G., Premecz G., Sain B., Mühlrád A. Selective carbethoxylation of the histidine residues of actin by diethylpyrocarbonate. Eur J Biochem. 1974 May 2;44(1):7–12. doi: 10.1111/j.1432-1033.1974.tb03452.x. [DOI] [PubMed] [Google Scholar]
  16. Holmquist B., Bünning P., Riordan J. F. A continuous spectrophotometric assay for angiotensin converting enzyme. Anal Biochem. 1979 Jun;95(2):540–548. doi: 10.1016/0003-2697(79)90769-3. [DOI] [PubMed] [Google Scholar]
  17. Kaiser E., Colescott R. L., Bossinger C. D., Cook P. I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem. 1970 Apr;34(2):595–598. doi: 10.1016/0003-2697(70)90146-6. [DOI] [PubMed] [Google Scholar]
  18. Koerber S. C., Fink A. L. The analysis of enzyme progress curves by numerical differentiation, including competitive product inhibition and enzyme reactivation. Anal Biochem. 1987 Aug 15;165(1):75–87. doi: 10.1016/0003-2697(87)90203-x. [DOI] [PubMed] [Google Scholar]
  19. Makinen K. K., Makinen P. L. Purification and properties of an extracellular collagenolytic protease produced by the human oral bacterium Bacillus cereus (strain Soc 67). J Biol Chem. 1987 Sep 15;262(26):12488–12495. [PubMed] [Google Scholar]
  20. Mäkinen K. K., Mäkinen P. L., Syed S. A. 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. J Biol Chem. 1992 Jul 15;267(20):14285–14293. [PubMed] [Google Scholar]
  21. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  22. Schmidt T. M., Bleakley B., Nealson K. H. Characterization of an Extracellular Protease from the Insect Pathogen Xenorhabdus luminescens. Appl Environ Microbiol. 1988 Nov;54(11):2793–2797. doi: 10.1128/aem.54.11.2793-2797.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Szállás E., Koch C., Fodor A., Burghardt J., Buss O., Szentirmai A., Nealson K. H., Stackebrandt E. Phylogenetic evidence for the taxonomic heterogeneity of Photorhabdus luminescens. Int J Syst Bacteriol. 1997 Apr;47(2):402–407. doi: 10.1099/00207713-47-2-402. [DOI] [PubMed] [Google Scholar]
  24. Van Wart H. E., Steinbrink D. R. A continuous spectrophotometric assay for Clostridium histolyticum collagenase. Anal Biochem. 1981 May 15;113(2):356–365. doi: 10.1016/0003-2697(81)90089-0. [DOI] [PubMed] [Google Scholar]
  25. Wee K. E., Yonan C. R., Chang F. N. A new broad-spectrum protease inhibitor from the entomopathogenic bacterium Photorhabdus luminescens. Microbiology. 2000 Dec;146(Pt 12):3141–3147. doi: 10.1099/00221287-146-12-3141. [DOI] [PubMed] [Google Scholar]
  26. Zimmerle C. T., Frieden C. Analysis of progress curves by simulations generated by numerical integration. Biochem J. 1989 Mar 1;258(2):381–387. doi: 10.1042/bj2580381. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES