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Nonchromogenic hydrolysis of elastase and cathepsin G p-nitroanilide substrates by Pseudomonas aeruginosa elastase
A Pelletier1, J L Dimicoli, C Boudier
1Laboratoire d'Enzymologie, INSERM Unité 237, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, Orsay, France.
Abstract:
Pseudomonas aeruginosa, which may cause severe lung infections, secretes a metalloelastase that may interfere with the assay of neutrophil elastase and cathepsin G in lung secretions. Using nuclear magnetic resonance spectroscopy, we have shown that P. aeruginosa elastase (PsE) cleaves succinyl-Ala3-p-nitroanilide between the first and the second alanine residue, rendering this substrate inefficient for the assay of neutrophil elastase. The cleavage occurs with a kcat/Km of 2.4 X 10(3) M-1 s-1, a value eightfold higher than the kcat/Km for the chromogenic cleavage of succinyl-Ala3-p-nitroanilide by neutrophil elastase. P. aeruginosa elastase also cleaves the elastase substrate succinyl-Ala3-Val-p-nitroanilide between the second and the third alanine residue and the cathepsin G substrate succinyl-Ala2-Pro-Phe-p-nitroanilide at the Pro-Phe linkage. By contrast, methoxysuccinyl-Ala2-Pro-Val-p-nitroanilide, another elastase substrate, is not hydrolyzed by the bacterial enzyme. Our data indicate that synthetic substrates should be used with caution to assay elastase and cathepsin G in lung secretions or other biologic fluids in which metalloproteinases may be present.
Insights
Pseudomonas aeruginosa elastase interferes with assays for neutrophil elastase and cathepsin G in lung secretions. This bacterial enzyme degrades common substrates, necessitating caution when analyzing these enzymes in biological fluids.
Area of Science:
- Biochemistry
- Microbiology
- Pulmonary Medicine
Background:
- Pseudomonas aeruginosa is a pathogen causing severe lung infections.
- Bacterial metalloelastases can interfere with host enzyme assays.
- Accurate quantification of neutrophil elastase and cathepsin G is crucial in respiratory conditions.
Purpose of the Study:
- To investigate the enzymatic activity of Pseudomonas aeruginosa elastase (PsE) on substrates used for neutrophil elastase and cathepsin G assays.
- To determine the specific cleavage sites and kinetics of PsE activity on these substrates.
- To assess the implications of PsE activity for diagnostic assays in lung secretions.
Main Methods:
- Nuclear magnetic resonance (NMR) spectroscopy was employed to analyze enzymatic reactions.
- Synthetic peptide substrates, including succinyl-Ala3-p-nitroanilide and succinyl-Ala2-Pro-Phe-p-nitroanilide, were used.
- Kinetic parameters (kcat/Km) were determined for substrate cleavage by PsE and compared to neutrophil elastase.
Main Results:
- PsE efficiently cleaves succinyl-Ala3-p-nitroanilide between the first and second alanine residues, rendering it unsuitable for neutrophil elastase assays.
- The cleavage rate (kcat/Km) of this substrate by PsE is eightfold higher than that by neutrophil elastase.
- PsE also degrades specific substrates for cathepsin G and another elastase substrate, succinyl-Ala3-Val-p-nitroanilide, but not methoxysuccinyl-Ala2-Pro-Val-p-nitroanilide.
Conclusions:
- Pseudomonas aeruginosa elastase significantly interferes with the accurate assay of neutrophil elastase and cathepsin G.
- The use of certain synthetic substrates for enzyme quantification in lung secretions containing PsE is unreliable.
- Caution is advised when interpreting results from neutrophil elastase and cathepsin G assays in the presence of metalloproteinases like PsE.