Abstract
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 Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 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. Vmax 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 Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax 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.
Highlights
The phosphotriesterase (PTE)1 from Pseudomonas diminuta has a rather broad substrate specificity
Construction, Expression, and Purification of Mutant Enzymes—The W131K, F132H, F132Y, F306E, F306H, F306K, and F306Y phosphotriesterase mutants were made using the method of overlap extension PCR
The isolated StyI/EcoRV restriction fragments from the F132H and F132Y mutants were substituted for the corresponding fragments within the already altered genes for the F306H and F306Y mutants
Summary
Materials—All chemicals were purchased from Sigma, Aldrich, Fisher, or U. The mutagenic PCR fragments were purified from an agarose gel, cut with BamHI, concentrated with BamHI-digested pBSϩ, ligated with T4 DNA ligase, and transformed into XL1-Blue cells. The mutant plasmids containing either the single tyrosine or histidine mutation at position 132 was digested with StyI and EcoRV to obtain a fragment containing the mutation at this site. This oligonucleotide fragment was ligated into an identically cut plasmid containing only the single mutation at position 306. Enzyme Assays and Data Analysis—The rate of paraoxon hydrolysis by each of the mutants was measured by monitoring the appearance of p-nitrophenol at 400 nm (⑀ ϭ 17,000 MϪ1 cmϪ1) at pH 9.0 [10]. The kinetic constants were obtained by a fit of the data to Equation 1 using the software supplied by Savanna Shell Software, where v ϭ velocity, V ϭ maximum velocity, A ϭ substrate concentration, and Ka is the Michaelis constant
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