Micellar electrokinetic chromatography for analyzing active site specificity of Pseudomonas aeruginosa elastase

The geometry of the catalytic site of Pseudomonas aeruginosa elastase was reexamined, exploiting the specific feature of micellar electrokinetic chromatography (MEKC), i.e., its ability to detect a decrease of intact substrate and simultaneous formation of reaction products. We carried out a detaile...

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Bibliographic Details
Published in:Electrophoresis 1999-06, Vol.20 (7), p.1578-1585
Main Authors: Viglio, Simona, Zanaboni, Giuseppe, Lupi, Anna, Gianelli, Luca, Luisetti, Maurizio, Casali, Lucio, Cetta, Giuseppe, Iadarola, Paolo
Format: Article
Language:English
Subjects:
Amino Acids - analysis
Binding Sites
Catalysis
Chromatography, Micellar Electrokinetic Capillary - methods
Kinetics
Mass Spectrometry
Micellar electrokinetic chromatography
Pancreatic Elastase - chemistry
Peptides - analysis
Protease active site
Pseudomonas aeruginosa - enzymology
Time Factors
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Summary:The geometry of the catalytic site of Pseudomonas aeruginosa elastase was reexamined, exploiting the specific feature of micellar electrokinetic chromatography (MEKC), i.e., its ability to detect a decrease of intact substrate and simultaneous formation of reaction products. We carried out a detailed investigation using two tri‐ and six tetra‐peptide 4‐nitroanilides (NA) differing from each other by only one or more amino acids as stable substrates. The kinetic cleavage parameters Km and kcat determined by MEKC and the catalytic efficiency Km/kcat values calculated allowed us to better define the substrate specificity of this proteinase.
ISSN:0173-0835
1522-2683
DOI:10.1002/(SICI)1522-2683(19990601)20:7<1578::AID-ELPS1578>3.0.CO;2-U