Stereochemical constraints on the substrate specificity of phosphotriesterase.
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abstract
A series of achiral, chiral, and racemic mixtures of paraoxon analogues containing various combinations of methyl, ethyl, isopropyl, or phenyl substituents were synthesized as probes of the stereochemical constraints within the active site of phosphotriesterase. The kinetic constants for these paraoxon analogues with the enzyme varied significantly with the size of substituents surrounding the phosphorus center. These results indicate that binding and catalysis depend significantly on the relative size and orientation of the two subsites that must accommodate the coordination of the alkyl or aryl substituents within the enzyme active site. Individual enantiomers of paraoxon analogues were also synthesized and the stereochemical specificity for phosphotriesterase determined. In general, the kinetic constants, kcat and kcat/Km, for the (-)-enantiomers of these phosphotriesters were 1-2 orders of magnitude greater than the (+)-enantiomers. In every case, the preferred isomer is of the SP-configuration. For example, the kcat/Km for SP-(-)-ethyl phenyl p-nitrophenyl phosphate is 1.8 x 10(8) M-1 s-1 but is only 1.8 x 10(6) M-1 s-1 for the RP-(+)-isomer. These results suggest that one enantiomer is positioned for hydrolysis more favorably than the other enantiomer. The inactivation of acetylcholinesterase with the same series of organophosphate nerve agents was also measured. The stereoisomer that more rapidly inactivates human acetylcholinesterase is hydrolyzed more slowly than its enantiomer by the phosphotriesterase.
