AUGMENTED HYDROLYSIS OF DIISOPROPYL FLUOROPHOSPHATE IN ENGINEERED MUTANTS OF PHOSPHOTRIESTERASE

The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide var iety of organophosphate insecticides and acetylcholinesterase inhibito rs. The rate of hydrolysis depends on the substrate and can range from 6000 s(-1) for paraoxon to 0.03 s(-1) for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotries terase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from act ive site residues in the wild type protein to a bound substrate analog were measured, and Trp(131), Phe(132), and Phe(306) were found to be located within 5.0 Angstrom of the oxygen atom of the leaving group, E leven mutants were created using site-directed mutagenesis and purifie d to homogeneity. Phe(132) and Phe(306) were replaced by tyrosine and/ or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were a lso constructed. Kinetic constants were measured for all of the mutant s with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. V-max values for the mutant enzymes with t he substrate paraoxon varied from near wild type values to a 4-order o f magnitude decrease for the W131K mutant, There were significant incr eases in the K-m for paraoxon for all mutants except F132H. V-max valu es measured using diethylphenylphosphate decreased for all mutants exc ept for F132H and F132Y, whereas K-m values ranged from near wild type levels to increases of 25-fold, V-max values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131R, K-m values for acephate ranged from near wild type to a 5-fold increas e. V-max values for the mutants tested with the substrate diisopropylf luorophosphate showed an increase in all cases except for the W131K, F 306K, and F306E mutants. The V-max value for the F132H/F306H mutant wa s increased to 3100 s(-1). These studies demonstrated for the first ti me that it is possible to significantly enhance the ability of the nat ive phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.