posted on 2020-03-17, 19:07authored byAndrew
N. Bigley, Tamari Narindoshvili, Dao Feng Xiang, Frank M. Raushel
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 extent of 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 were 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.