Nonchromogenic hydrolysis of elastase and cathepsin G p-nitroanilide substrates by Pseudomonas aeruginosa elastase.
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.
Pelletier
A
A
Laboratoire d'Enzymologie, INSERM Unité 237, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, Orsay, France.
Dimicoli
J L
JL
Boudier
C
C
Bieth
J G
JG
D000595:Amino Acid Sequence; D000814:Aniline Compounds; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D000814:Aniline Compounds; C019498:4-nitroaniline; D002403:Cathepsins; D012697:Serine Endopeptidases; D056649:Cathepsin G; D010196:Pancreatic Elastase