Rationally Engineered Mutants of Phosphotriesterase for Preparative Scale Isolation of Chiral Organophosphates

Abstract

will catalyze the hydrolysis of the most acidic phenolic substituent from an organophosphate triester. The overall rate of hydrolysis is very much dependent on the pKa of the leaving group phenol. 4b To enhance the substrate stereoselectivity exhibited by the wildtype enzyme, we designed and characterized several site-directed mutants of PTE. Three regions within the active site of PTE (small, large, and leaVing group) have been identified that interact with the three substituents attached to the phosphorus center of typical substrates. 7 These subsites were graphically localized using the X-ray crystal structure of PTE in the presence of a bound substrate analogue. The relative sizes of these binding subsites appear to play the dominant roles in establishing the stereoselective properties of the wild-type PTE. 8 Thus, when the small subsite is reduced in size by mutating Gly60 to an alanine residue, the observed stereoselectivity of the mutant enzyme is dramatically enhanced toward the hydrolysis of the SP-isomer. For the racemic pairs of substrates I, II, and III, the kcat/Km values are 10- to 400-fold greater for the SP-isomers than for the corresponding RP-isomers and 10 000- to 15 000-fold greater for the