The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. 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 phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active 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, Eleven mutants were created using site-directed mutagenesis and purified 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 also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. V-max values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant, There were significant increases in the K-m for paraoxon for all mutants except F132H. V-max values measured using diethylphenylphosphate decreased for all mutants except 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 increase. V-max values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The V-max value for the F132H/F306H mutant was increased to 3100 s(-1). These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.