Structural Determinants of the Substrate and Stereochemical Specificity of Phosphotriesterase

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Abstract
Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the SP-enantiomer over the corresponding RP-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased kcat and kcat/Ka for the RP-enantiomers, but had little influence on the kinetic constants for the SP-enantiomers of the chiral substrates. As a result, the chiral preference for the SP-enantiomers was greatly enhanced. For example, the value of kcat/Ka with the mutant G60A for the SP-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding RP-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the SP-enantiomers, due to selective increases in the reaction rates for the RP-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on kcat/Ka for either the RP- or SP-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.