Kinetics of ampicillin synthesis catalyzed by penicillin acylase from E. coli in homogeneous and heterogeneous systems. Quantitative characterization of nucleophile reactivity and mathematical modeling of the process.
Kinetic regularities of the enzymatic acyl group transfer reactions have been studied using ampicillin synthesis catalyzed by E. coli penicillin acylase as an example. It was shown that ampicillin synthesis proceeds through the formation of an acylenzyme-nucleophile complex capable of undergoing hydrolysis. The relative nucleophile reactivity of 6-aminopenicillanic acid (6-APA) is a complex parameter dependent on the nucleophile concentration. The kinetic analysis showed that the maximum yield of antibiotic being synthesized depended only on the nucleophile reactivity of 6-APA, the ratio between the enzyme reactivities with respect to the target product and acyl donor, and the initial concentrations of reagents. The parameters characterizing the nucleophile reactivity of 6-APA have been determined. The algorithm of modeling the enzymatic synthesis has been elaborated. The proposed algorithm allows the kinetics of the process not only in homogeneous, but also in heterogeneous ("aqueous solution-precipitate") systems to be quantitatively predicted and described based on experimental values of parameters of the reaction. It was shown that in heterogeneous "aqueous solution-precipitate" systems PA-catalyzed ampicillin synthesis proceeds much more efficiently compared to the homogeneous solution.
Youshko
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MI
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia.
Svedas
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VK
D000667:Ampicillin / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008962:Models, Theoretical; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D010399:Penicillin Amidase / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
C033393:methyl phenylglycine; C008852:2-phenylglycine; D000667:Ampicillin; D010397:Penicillanic Acid; D010399:Penicillin Amidase; C004408:aminopenicillanic acid; D005998:Glycine