Study of nucleophile binding in the penicillin acylase active center. Kinetic analysis.
The influence of the external nucleophile (6-aminopenicillanic acid) on the kinetics of the penicillin acylase-catalyzed acyl transfer reactions was studied using a highly sensitive spectrophotometric assay. An adequate kinetic scheme is suggested based on kinetic analysis of the experimental dependencies of the k(cat) and K(m) values on the nucleophile concentration. The proposed kinetic scheme has been verified by a quantitative description of the above-mentioned experimental dependencies using the set of kinetic parameters obtained from independent experiments. Such an approach can be used for modeling of different penicillin acylase-catalyzed acyl transfer reactions.
Youshko
M I
MI
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
Bukhanov
A L
AL
Svedas
V K
VK
D062365:Aminobenzoates / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D010399:Penicillin Amidase / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity
D062365:Aminobenzoates; D029968:Escherichia coli Proteins; D010397:Penicillanic Acid; D010399:Penicillin Amidase; C004408:aminopenicillanic acid