Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens.
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.
Marokházi
Judit
J
Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C, Budapest, H-1117, Hungary.
Kóczán
György
G
Hudecz
Ferenc
F
Gráf
László
L
Fodor
András
A
Venekei
István
I
D001920:Bradykinin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D013058:Mass Spectrometry; D061965:Matrix Metalloproteinase Inhibitors; D008670:Metals / Q000494:pharmacology; D020637:Photorhabdus / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D012984:Software; D013379:Substrate Specificity
D061965:Matrix Metalloproteinase Inhibitors; D008670:Metals; D011480:Protease Inhibitors; D006639:Histidine; D005780:Gelatin; D003094:Collagen; D017364:Collagenases; D003545:Cysteine; D001920:Bradykinin