Structural determinants of the substrate and stereochemical specificity ofphosphotriesterase

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide varie ty of organophosphate nerve agents and insecticides. Previous kinetic studi es with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the Sp-enantiomer over the correspondin g R-p-enantiomers by factors ranging from 1 to 90. The three-dimensional cr ystal structure of PTE with a bound substrate analogue has led to the ident ification of three hydrophobic binding pockets. To delineate the factors th at govern the reactivity and stereoselectivity of PTE, the dimensions of th ese three subsites have been systematically altered by site-directed mutage nesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-25 7, 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 sub site dramatically decreased k(cat) and k(cat)/K-a for the R-p-enantiomers, but had little influence on the kinetic constants for the S-p-enantiomers o f the chiral substrates. As a result, the chiral preference for the Sp-enan tiomers was greatly enhanced. For example, the value of k(cat)/K-a 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 s mall or leaving group subsites, caused a significant reduction in the enant iomeric preference for the Sp-enantiomers, due to selective increases in th e reaction rates for the Rp-enantiomers. Enlargement of the large subsite b y the construction of an H254A, H257A, L271A, or M317A mutant had a relativ ely small effect on k(cat)/K-a for either the R-p- or S-p-enantiomers and t hus had little effect on the overall stereoselectivity. These studies demon strate that by modifying specific residues located within the active site o f PTE, it is possible to dramatically alter the stereoselectivity and overa ll reactivity of the native enzyme toward chiral substrates.