Organophosphate Triesters Research Articles

Hydrolysis of organophosphate triesters by Escherichia coli aminopeptidase P

Aminopeptidase P (AMPP, EC 3.4.11.9) from Escherichia coli catalyzes the cleavage of amino-terminal X-Pro peptide bonds. In this study, AMPP was found to catalyze the hydrolysis of a wide range of organophosphate triesters ( 1– 7). The activity of AMPP was promoted in the presence of Mn 2+, and slight activation was achieved by treatment with Cd 2+. The enzymatic rates of hydrolysis for organophosphate triesters ( 3 and 5) with both isopropyl (or isobutyl) and methyl substituents attached to the phosphorus atom were from 5- to 36-fold higher compared to the corresponding analogs ( 4 and 6, 7). Three mutants, R153W, R153L and R370L, have been characterized and exhibited from 1.2- to 1.5-fold increases in hydrolyzing 1– 7 compared to the wild-type enzyme. These results demonstrate that there is a flanking hydrophobic interaction between the substituent group of substrates and position 153 or 370 in AMPP to facilitate the hydrolysis of organophosphate triesters.

Miniaturized dynamic liquid-liquid extraction of organophosphate triesters from blood plasma using the hollow fibre-based XT-tube extractor.

A miniaturized liquid-liquid extractor for bioanalytical sample preparation is described. The extractor consists of a polypropylene hollow fibre mounted inside polytetrafluoroethylene (PTFE) tubing by means of a cross (X) connector and a tee (T) connector. All parts are commercially available, inexpensive, and easily assembled. The aqueous sample, injected into a carrier flow, is pumped along the outside of the fibre and the organic phase, which also wets the pores of the hollow fibre wall, is pumped inside. Eight organophosphate triester (OPE) plasticisers/flame retardants were extracted from 50 microL spiked blood plasma that had been mixed with 50 microL formic acid to denature plasma proteins. The organic phase was a mixture of hexane and methyl tert-butyl ether (MTBE). A high concentration of formic acid in the sample and of MTBE in the organic phase had positive effects on the recovery of some OPE. When investigating the recovery as a function of extraction time it was found that the extraction reached a maximum after 10 min, at a flow-rate of 15 microL min(-1). Recoveries varied between 40 and 80% with RSD around 4% for most compounds. The whole 150- microL extract was injected into a GC-MS system equipped with a programmed-temperature vaporization (PTV) injector. With the MS in selected-ion monitoring (SIM) mode, the LOD for triphenyl phosphate and 2-ethylhexyl diphenyl phosphate were 0.3 and 0.2 ng mL(-1), respectively. More than 40 plasma extractions were performed with the same fibre without any detectable change in extraction efficiency.

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase.

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, 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 subsite 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 of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.

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

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

Improvement of enantioselectivity of chiral organophosphate insecticide hydrolysis by bacterial phosphotriesterase.

The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp. can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be an effective catalyst for the degradation of toxic organophosphate esters. Ethyl 4-nitrophenyl phenylphosphonothioate (EPN) is a chiral organophosphate. Optical isomers of EPN show differences in their toxicity. R-EPN is known to be more toxic to hens and houseflies than S-EPN. We determined the Ki value of each enantiomer toward electric eel acetylcholinesterase. R-EPN (Ki = 6 microM) inhibited acetylcholinesterase much more effectively than S-EPN (Ki = 52 microM) did in vitro. Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively. When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration. At 20% DMSO, the maximum activity was observed. The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed. The Km value for R-EPN decreased from 0.24 to 0.03 mM, and the Vmax value increased from 0.25 to 0.60 U/mg of protein. Vmax/Km values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.

Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10(8) times faster than the uncatalyzed reaction (kcat = 0.06 s-1 and Km = 38 mM). Upon the addition of various alkylamines to the reaction mixture, the kcat/Km for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (kcat = 0.34 s-1 and Km = 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wild-type enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.

Utilization of Copper as a Paramagnetic Probe for the Binuclear Metal Center of Phosphotriesterase

Bacterial phosphotriesterase catalyzes the hydrolysis of organophosphate triesters. To be active, the enzyme requires that two divalent cations are bound. These metal ions are bound in close proximity to one another as a binuclear center. To characterize the structure and function of the binuclear metal binding sites, we have prepared the copper-substituted enzyme. The kinetic data indicate that this enzyme is essentially inactive toward the hydrolysis of phosphotriesters. The EPR signal arising from the copper-substituted enzyme is nearly axial, with g|| = 2.24 and g⊥ = 2.05 and shows at least seven superhyperfine transitions in the g⊥ region with A⊥ = 1.45 × 10−3 cm−1. These splittings are consistent with the direct ligation of more than one nitrogen to the metal center. The average spin quantitation of copper-substituted enzymes are 0.6 spin/Cu, approximately half of that observed for noninteracting Cu2+ ions. The spin intensity increases to ca. 1 spin/Cu when samples are denatured with acid. The binding of metal ions to the designated α and β sites is highly synergistic (i.e., the metal ions bind in pairs). Mixed metal complexes of the type Cu/X and X/Cu were prepared. When X is a diamagnetic ion (Zn2+ or Cd2+), the spin quantitation increases, but when X is the paramagnetic Co2+ ion, the spin quantitation decreases. This behavior indicates that the low spin intensities observed for copper-substituted phosphotriesterase arise from spin-coupling of the two adjacent Cu2+ ions. The addition of dithiothreitol, ascorbate, or dithionite to the copper-substituted phosphotriesterase results in nearly the complete loss of spin intensity. This indicates that the bound coppers can be reduced to the cuprous state.

Stereospcific enzymatic hydrolysis of phosphorus-sulfur bonds in chiral organophosphate triesters

The bacterial phosphotriesterase has been shown to be capable of catalyzing the hydrolysis of a P-S bond in organophosphate triesters. The V max values for the hydrolysis of the insecticides, acephate (O, S-dimethyl N-acetlphosphoramidothioate) and methamidophos (O, S-dimethyl phosphoramidothioate) are 140 and 6 min −1, respectively. The (S P)-enantiomers are hydrolyzed at a rate that is 100-fold faster than the (R P)-enantiomers.

ChemInform Abstract: Mono and Dinuclear M2+ Chelates as Catalysts for the Hydrolysis of Organophosphate Triesters.

AbstractThe 2:1 and 1:1 Zn(II) and Cu(II) complexes of the ligands (II) and (III), respectively, formed in solution are studied as catalysts for the hydrolysis of tris(4‐nitrophenyl)phosphate (I) in 33% EtOH‐H2.

Mono and dinuclear M 2+ chelates as catalysts for the hydrolysis of organophosphate triesters

N,N,N′,N′-Tetrakis(1-methylimidazol-2-ylmethyl)pentane-1,5-diamine ( 1) and bis(1-methylimidazol-2-ylmethyl)ethylamine ( 2) were prepared and their Zn 2+ and Cu 2+ complexes studied as catalysts of the hydrolysis of tris-4-nitrophenyl phosphate ( 3) in 33% ethanol-water. A M 2+-bound H 2O undergoes ionization in all the 1: 2M 2+ and 2:M 2+ complexes. With 1:2Zn 2+ and 2:Zn 2+, both the acidic and the basic species promote the hydrolysis of 3, as do their corresponding Cu 2+ complexes. There is no evidence of a pre-equilibrium formation of ternary 3:L: nM 2+ complexes, so the basic forms of the complexes are acting as bimolecular general bases or nucleophiles towards 3. The observed order of activity ( 2:Cu 2+ > 2:Zn 2+ > 1:2Cu 2+ > 1:2Zn 2+) is a result of the nucleophilicity/basicity of the M 2+-bound OH − decreasing as the p K a decreases.