Stereoselective Hydrolysis of Organophosphate Nerve Agents by the Bacterial Phosphotriesterase

Abstract

Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well known are GA, GB, GD, GF, VX and VR. All of these compounds contain a chiral phosphorus center with the S P -enantiomers being significantly more toxic than the R P -enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R P -enantiomers. A series of enantiomerically pure chiral nerve agent analogues has been developed containing the relevant phosphoryl centers found in GB, GD, GF, VX and VR. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed stereoselectivity, have been identified. A number of variants showed dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S P -enantiomers. Improvements of up to three orders of magnitude relative to the wild type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.Organophosphorus compounds have been utilized for more than 50 years as insecticides for the protection of agricultural crops (1) and similar compounds have been developed as chemical warfare agents (2). The structures of these latter compounds are presented in Scheme 1 and include tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX and VR. GA has a cyanide leaving group, the three remaining G-agents (GB, GD, and GF) have a fluoride leaving group, and the two versions of VX have a thiolate leaving group. The toxicity of these organophosphonates is due to the inactivation of acetylcholinesterase (AChE), an enzyme that catalyzes the hydrolysis of acetylcholine at neural synapses, through the phosphonylation of an active site serine residue (3). GA, GB, GF, VX, and VR contain a chiral phosphorus center and thus each of these nerve agents has two stereoisomers, while soman has four stereoisomers because of an additional chiral center within the pinacolyl substituent. The enantiomers are differentially toxic; the S Pstereoisomer of sarin reacts with AChE approximately ~10 4 times faster than the R Pstereoisomer and the two S P -stereoisomers of soman react ~10 5 times faster than the two † This work was supported by the NIH (GM 68550).

Function Discovery and Structural Characterization of a Methylphosphonate Esterase

“…This stereoselectivity is opposite to that observed for wild-type PTE (33). The organophosphate and organophosphonate esters are toxic because of their ability to rapidly inactivate the enzyme acetyl cholinesterase.…”

“…From the time course for the hydrolysis of racemic 6 by PTE alone (Figure 4B) and Pmi1525 alone (Figure 4C) it can be concluded that Pmi1525 and PTE preferentially hydrolyze the opposite enantiomers from one another. Since PTE hydrolyzes the ( R P )-enantiomer of isobutyl 4-acetylphenyl methylphosphonate ( 9 ) 25 times faster than the ( S P )-enantiomer ( 10 ), Pmi1525 prefers to hydrolyze the ( S P )-enantiomer of this substrate (33). For wild-type PTE, the ( R P )-enantiomer of compound 6 is hydrolyzed 75 times faster than the ( S p )-enantiomer based on a fit of the data in Figure 4B to the sum of two exponentials (29).…”

Variants of Phosphotriesterase for the Enhanced Detoxification of the Chemical Warfare Agent VR

“…7,22 The individual enantiomers of p-acetophenyl VR (APVR) were synthesized as previously described. 16 Samples of VX and VR were Chemical Agent Standard Analytical Reference Material of the 8 highest purity available, and were further purified as described previously. 7 Unless otherwise noted, all other chemicals were purchased from Sigma Aldrich.…”

“…Wild-type PTE has enzymatic activity for the hydrolysis of racemic VR similar to VX, but the larger isobutyl group attached to the phosphorus center results in a 25-fold preference for the RPenantiomer. 16,19 The catalytic activity of wild-type PTE for the hydrolysis of the SPenantiomer of VR (kcat/Km = 4.3 M -1 s -1 ) is significantly lower than for the hydrolysis of VX.…”

“…Wild-type PTE is known to prefer to hydrolyze the RP-enantiomer of the VR chiral center, while QF is known to prefer the SP-enantiomer of VX 7,16. .…”