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Detection of Protease Activity by Fluorescent Peptide Zymography
Published on: January 20, 2019
Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium,
Judit Marokházi1, György Kóczán, Ferenc Hudecz
1Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C, Budapest, H-1117, Hungary.
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.
Insights
Photorhabdus protease B (Php-B), a novel metalloprotease from Photorhabdus luminescens, functions as a collagen peptidase. This enzyme, dependent on histidine, cysteine, and Zn2+, degrades collagen fragments, aiding bacterial nutrition.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Photorhabdus luminescens is an entomopathogenic bacterium.
- Proteolytic enzymes play crucial roles in bacterial pathogenesis and nutrition.
Purpose of the Study:
- To purify and characterize a novel proteolytic enzyme, Php-B, from Photorhabdus luminescens.
- To elucidate the substrate specificity, catalytic mechanism, and potential biological role of Php-B.
Main Methods:
- Enzyme purification and mass determination.
- Substrate hydrolysis assays using various peptides and oligopeptides.
- Enzyme inhibition studies and metal ion dependency tests.
- Kinetic analysis using a numerically integrated Michaelis-Menten model.
Main Results:
- Php-B is an intracellular metalloprotease (74 kDa) active at pH 7.0.
- It exhibits significant hydrolytic activity against bradykinin and Fua-LGPA, with kcat=3.6x10(2) s(-1) and K(m)=5.8x10(-5) M(-1).
- Enzyme activity depends on histidine and cysteine residues and requires Zn2+.
- Php-B cleaves collagen-like sequences but not native collagens or gelatin, identifying it as a collagen peptidase.
Conclusions:
- Php-B is the first identified collagen peptidase from an insect pathogen.
- This enzyme likely contributes to the nutrition of Photorhabdus luminescens by degrading collagen fragments.
- Numerical integration of Michaelis-Menten kinetics provides a reliable method for characterizing collagen-peptide-hydrolyzing enzymes.
