Stereoselective Formation of Multiple Reaction Products by the Phosphotriesterase from Sphingobium sp. TCM1

The organophosphate flame-retardants are used to inhibit combustion and increase plasticity in plastics and durable foams. While not neurotoxic, these compounds are potential carcinogens, endocrine disrupters and developmental toxins. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is unique among phosphotriesterase enzymes for its ability to hydrolyze these compounds, and its ability to hydrolyze any one of the three different ester bonds within a given substrate. In some cases, the hydrolysis of a methyl ester exceeds that of a p-nitrophenyl ester within a single substrate. There is a stereochemical component to this hydrolysis where the two enantiomers of chiral substrates give different product ratios. To investigate the stereoselectivity for the product distribution of Sb-PTE, a series of 24 phosphotriesters was synthesized with all possible combinations of methyl, cyclohexyl, phenyl and p-nitrophenyl esters. Prochiral compounds were made chiral by differential isotopic labeling using a chemo/enzymatic strategy, which allowed the differentiation of hydrolysis for each ester in all but two compounds. The rate equations for this unique enzymatic mechanism were derived, the product ratios were determined for each substrate, and the individual kinetic constants for hydrolysis of each ester within each substrate were measured. The findings are consistent with the rate limiting step for substrate hydrolysis catalyzed by Sb-PTE being the formation of a phosphorane-like intermediate and the kinetic constants and product ratios being dictated by a combination of transition state energies, inductive effects, and stereochemical constraints.