Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase

• Published in: Biochemistry (Easton) 1988-03, Vol.27 (5), p.1591-1597
• Main Authors: Lewis, Vincent E, Donarski, William J, Wild, James R, Raushel, Frank M
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemical Phenomena; Chemistry; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Organothiophosphorus Compounds - metabolism; Phosphoric Monoester Hydrolases - metabolism; Pseudomonas - enzymology; Pseudomonas diminuta; Stereoisomerism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00405a030

The reaction mechanism for the phosphotriesterase from Pseudomonas diminuta has been examined. When paraoxon (diethyl 4-nitrophenyl phosphate) is hydrolyzed by this enzyme in oxygen-18-labeled water, the oxygen-18 label is found exclusively in the diethyl phosphate product. The absolute configurations for the (+) and (-) enantiomers of O-ethyl phenylphosphonothioic acid have been determined by X-ray diffraction structural determination of the individual crystalline 1-phenylethylamine salts. The (+) enantiomer of the free acid corresponds to the RP configuration. The RP enantiomer of O-ethyl phenylphosphonothioic acid has been converted to the SP enantiomer of EPN [O-ethyl O-(4-nitrophenyl) phenylphosphonothioate]. (SP)-EPN is hydrolyzed by the phosphotriesterase to the SP enantiomer of O-ethyl phenylphosphonothioic acid. The enzymatic reaction therefore proceeds with inversion of configuration. These results have been interpreted as an indication of a single in-line displacement by an activated water molecule directly at the phosphorus center of the phosphotriester substrate. (RP)-EPN is not hydrolyzed by the enzyme at an appreciable rate.