Phosphorus of the Reaction Catalyzed by a Bacterial Phosphotriesteraset

The reaction mechanism for the phosphotriesterase from Pseudomonas diminuta has been examined. When paraoxon (diethyl 4-nitrophenyl phosphate) is hydrolyzed by this enzyme in oxygen18-labeled water, the oxygen-18 label is found exclusively in the diethyl phosphate product. The absolute configurations for the (+) and (-) enantiomers of 0-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 0-ethyl phenylphosphonothioic acid has been converted to the Sp enantiomer of E P N [ 0-ethyl 0-(4-nitrophenyl) phenylphosphonothioate] . (Sp)-EPN is hydrolyzed by the phosphotriesterase to the Sp enantiomer of 0-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 a t the phosphorus center of the phosphotriester substrate. (Rp)-EPN is not hydrolyzed by the enzyme a t an appreciable rate. A broad variety of organophosphate triesters have been used as pesticides, insecticides, and other biological control agents. First discovered in Germany in the 1930s (Schrader, 1963), 'This work was supported by the Army Research Office (DAAL0387-K-0017) and the Robert A. Welch Foundation (A-840 and A-915). F.M.R. is the recipient of NIH Research Career Development Award DK-01366. W.J.D. is the recipient of an IUCCP fellowship from Texas A & M University. The authors acknowledge support by the Board of Regents of Texas A & M University. *Address correspondence to this author at the Department of Chemistry, Texas A & M University. some modern organophosphates are considered to be among the safest of all chemical pesticides. However, many compounds known to be mammalian neurotoxins (McEwen & Stephenson, 1979) are also included in this class of compounds. In general, these pesticides are considered to be chemically reactive materials that do not persist in the soil for long periods of time. The lack of persistence in the soil has been attributed to their susceptibility to microbial hydrolysis (Munnecke, 198 1). The microbial degradation of organophosphates such as paraoxon (diethyl 4-nitrophenyl phosphate) or the related phosphorothioate (e.g., parathion) has been defined in several 0006-2960/88/0427-1591$01.50/0