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

Biochem J. 2004 May 1;379(Pt 3):633-40. doi: 10.1042/BJ20031116.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bradykinin / metabolism
  • Chromatography, High Pressure Liquid
  • Collagen / metabolism
  • Collagenases / chemistry
  • Collagenases / isolation & purification*
  • Collagenases / metabolism*
  • Cysteine / metabolism
  • Gelatin / metabolism
  • Histidine / metabolism
  • Hydrogen-Ion Concentration
  • Hydrolysis / drug effects
  • Kinetics
  • Mass Spectrometry
  • Matrix Metalloproteinase Inhibitors
  • Metals / pharmacology
  • Photorhabdus / enzymology*
  • Protease Inhibitors / pharmacology
  • Software
  • Substrate Specificity


  • Matrix Metalloproteinase Inhibitors
  • Metals
  • Protease Inhibitors
  • Histidine
  • Gelatin
  • Collagen
  • Collagenases
  • Cysteine
  • Bradykinin