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

Judit Marokházi, György Kóczán, F. Hudecz, L. Gráf, A. Fodor, I. Venekei

Research output: Contribution to journalArticle

13 Citations (Scopus)

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, FuaLGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat = 3.6 × 102 s-1, Km = 5.B × 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.

Original languageEnglish
Pages (from-to)633-640
Number of pages8
JournalBiochemical Journal
Volume379
Issue number3
DOIs
Publication statusPublished - May 1 2004

Fingerprint

Photorhabdus
Collagenases
Bacteria
Peptide Hydrolases
Oligopeptides
Enzymes
Collagen
Bradykinin
Peptides
Substrates
Enzyme kinetics
Ions
Collagen Type IV
Enzyme activity
Molecular mass
Metalloproteases
Enzyme Inhibitors
Pathogens
Gelatin
Nutrition

Keywords

  • Collagen peptidase
  • Metallopeptidase
  • Numerical simulation
  • Photorhabdus luminescens
  • Progress curve analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens. / Marokházi, Judit; Kóczán, György; Hudecz, F.; Gráf, L.; Fodor, A.; Venekei, I.

In: Biochemical Journal, Vol. 379, No. 3, 01.05.2004, p. 633-640.

Research output: Contribution to journalArticle

@article{29258fcaf42e40e98feaa0b9d34c1b99,
title = "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, FuaLGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat = 3.6 × 102 s-1, Km = 5.B × 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.",
keywords = "Collagen peptidase, Metallopeptidase, Numerical simulation, Photorhabdus luminescens, Progress curve analysis",
author = "Judit Marokh{\'a}zi and Gy{\"o}rgy K{\'o}cz{\'a}n and F. Hudecz and L. Gr{\'a}f and A. Fodor and I. Venekei",
year = "2004",
month = "5",
day = "1",
doi = "10.1042/BJ20031116",
language = "English",
volume = "379",
pages = "633--640",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "3",

}

TY - JOUR

T1 - Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens

AU - Marokházi, Judit

AU - Kóczán, György

AU - Hudecz, F.

AU - Gráf, L.

AU - Fodor, A.

AU - Venekei, I.

PY - 2004/5/1

Y1 - 2004/5/1

N2 - 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, FuaLGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat = 3.6 × 102 s-1, Km = 5.B × 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.

AB - 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, FuaLGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat = 3.6 × 102 s-1, Km = 5.B × 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.

KW - Collagen peptidase

KW - Metallopeptidase

KW - Numerical simulation

KW - Photorhabdus luminescens

KW - Progress curve analysis

UR - http://www.scopus.com/inward/record.url?scp=2542577762&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2542577762&partnerID=8YFLogxK

U2 - 10.1042/BJ20031116

DO - 10.1042/BJ20031116

M3 - Article

VL - 379

SP - 633

EP - 640

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -