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

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.

Proteolytic Enzyme Production by Strains of the Insect Pathogen Xenorhabdus and Characterization of an Early-Log-Phase-Secreted Protease as a Potential Virulence Factor

“…The substrates were purchased from Sigma-Aldrich (St. Louis, MO) (succinyl-Ala-Ala-Pro-Phe-thiobenzyl [Succ-AAPF-SBzl]) and Bachem (Bubendorf, Switzerland) (Fua-Leu-Gly-Pro-Ala [Fua-LGPA]) or prepared as described previously (succinyl-Ala-Ala-Pro-Phe-Lys-aminomethyl-coumarine [Succ-AAPX-AMC] [X is Phe and Lys] [13] and Fua-ALVY [16]). For solutions, the substrates were dissolved in dimethylformamide.…”

Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes

“…Php-D was separated during Php-B purification in a Sephadex G-100 gel filtration step (for details see reference 20). Fractions with the highest Fua-ALVY-ase activity (at an elution volume of 120 to 140 ml from a column [100 cm by 16 mm]) were pooled and concentrated to a volume of 3 ml by using a Centricon concentrator (Millipore).…”

“…In the case of Fua-blocked substrates, the decrease in absorbance at 324 nm was monitored until the end of the reaction. The catalytic activity (k obs ) was calculated by fitting the final portion of the curves (where the remaining substrate concentration was less than 1/10 the K m [ϳ40 M for Php-B] [20]) to first-order kinetics by using the Origin 5.0 software (Microcal).…”

Model-based experimental analysis of enzyme kinetics in aqueous–organic biphasic systems

“…An alternative to both approaches for studying enzyme kinetics is the use of dynamic parameter estimation, where the kinetic law is repeatedly integrated numerically (Bates and Frieden, 1973;Marokhazi et al, 2004). The advantage is that neither the rate equation nor the concentration data have to be modified by the user and statistically sound results can be obtained.…”