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 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 k(cat) determined by MEKC and the catalytic efficiency Km/k(cat) values calculated allowed us to better define the substrate specificity of this proteinase.
Viglio
S
S
Dipartimento di Biochimica A. Castellani, Università di Pavia, Italy.
Zanaboni
G
G
Lupi
A
A
Gianelli
L
L
Luisetti
M
M
Casali
L
L
Cetta
G
G
Iadarola
P
P
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002384:Catalysis; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D007700:Kinetics; D013058:Mass Spectrometry; D010196:Pancreatic Elastase / Q000737:chemistry*; D010455:Peptides / Q000032:analysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013997:Time Factors
D000596:Amino Acids; D010455:Peptides; D010196:Pancreatic Elastase