Stereochemical preferences for chiral substrates by the bacterial phosphotriesterase.

The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate nerve agents such as paraoxon (diethyl p-nitrophenyl phosphate) with a turnover number of approximately 10(4) s(-1). The active site of the enzyme has been shown to be composed of a binuclear Zn2+ complex with a bridging hydroxide. The utilization of chiral phosphotriesters has demonstrated that the overall hydrolytic reaction occurs with net inversion of stereochemistry at the phosphorus center. The stereochemical constraints of the active site have been probed by the synthesis and characterization of paraoxon analogs. One or both of the two ethoxy substituents of paraoxon have been replaced with various combinations of methyl, isopropyl, or phenyl groups. Racemic mixtures and individual enantiomers were tested as substrates for the phosphotriesterase. In general, the kinetic constants (k(cat) and k(cat)/Km) for the (-)-enantiomers were one to two orders of magnitude greater than the (+)-enantiomer. Conversely, acetylcholinesterase was more rapidly inactivated by the (+)-enantiomers than the (-)-enantiomers. These results were examined in the context of the three-dimensional structure of the bacterial phosphotriesterase.