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=3.6×102 s−1, Km=5.8×10−5 M−1, pH optimum approx. 7.0). The pKa1 and the pKa2 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.
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Research Article|
May 01 2004
Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens
Judit MAROKHÁZI;
Judit MAROKHÁZI
*Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C, Budapest, H-1117 Hungary
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György KÓCZÁN;
György KÓCZÁN
†Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest, Hungary
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Ferenc HUDECZ;
Ferenc HUDECZ
†Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest, Hungary
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László GRÁF;
László GRÁF
*Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C, Budapest, H-1117 Hungary
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András FODOR;
András FODOR
‡Department of Genetics, Eötvös Loránd University, Pázmány sétány 1/C, Budapest, H-1117 Hungary
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István VENEKEI
István VENEKEI
1
*Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C, Budapest, H-1117 Hungary
1To whom correspondence should be addressed (e-mail venekei@cerberus.elte.hu).
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Publisher: Portland Press Ltd
Received:
July 24 2003
Revision Received:
January 14 2004
Accepted:
January 26 2004
Accepted Manuscript online:
January 26 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 379 (3): 633–640.
Article history
Received:
July 24 2003
Revision Received:
January 14 2004
Accepted:
January 26 2004
Accepted Manuscript online:
January 26 2004
Citation
Judit MAROKHÁZI, György KÓCZÁN, Ferenc HUDECZ, László GRÁF, András FODOR, István VENEKEI; Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens. Biochem J 1 May 2004; 379 (3): 633–640. doi: https://doi.org/10.1042/bj20031116
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