Stereoselectivity toward VX Is Determined by Interactions with Residues of the Acyl Pocket as Well as of the Peripheral Anionic Site of AChE

Abstract

The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.

Crystal structures of oxime-bound fenamiphos-acetylcholinesterases: Reactivation involving flipping of the His447 ring to form a reactive Glu334–His447–oxime triad

“…The human enzyme was used to compare with a previous report [30] and due to similar reactivation kinetics of human and rat AChEs [31], high similarity among human, rat and mouse AChEs (89% sequence identity between the human and rat enzymes; 98% sequence identity between the rat and mouse enzymes), and the same active-site residues in the three enzymes. Compared to wild type, the inhibitions of Phe295Ala and Phe297Ala by E1 increased by ~3 fold (k i = 45.2  1.110 3 M -1 min -1 ) and decreased by ~5 fold (k i = 3.5  0.210 3 M -1 min -1 ), respectively.…”

“…In the present study, inhibitions of wild type hAChE, Phe295Ala and Phe297Ala by separated fenamiphos enantiomers show that none of the two mutants reverses the stereochemical preference. Moreover, for the more reactive enantiomer, the Phe295Ala mutation increases the inhibition constant by ~3 fold whereas the Phe297Ala On the other hand, inhibition kinetics of the phosphates ethylparaoxon, diethylfluorophosphate (DEFP) and echothiophate show a 20-40 fold enhanced inhibition potency in the Phe295Ala mutant [30,36]. One obvious difference between the methyl phosphonates discussed above and these phosphates is that the methyl phosphonates have methyl and ethoxy substituents whereas the phosphates have two ethoxy substituents.…”

“…It has been suggested that an ethoxy group may fit the acyl pocket without perturbing the conformations of Phe295 and Phe297 found in the apo mAChE structure [30]. This proposal was based on two observations of conjugates with one methyl-and one ethoxy substituent; i) inhibition kinetics where an increased size of the acyl pocket (Phe295Ala) had a small effect (~1.5 fold) on the reactivity for the R stereoisomer of non-charged O-ethyl S-3-(isopropyl-4-methylpentyl) methylphosphonothioate (P[R] ncVX) which has the ethoxy moiety towards the acyl pocket [30] and ii) the lack of stereoselectivity for MEPQ (7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide), which has a methyl-and an ethoxy substituent [35].…”

“…This proposal was based on two observations of conjugates with one methyl-and one ethoxy substituent; i) inhibition kinetics where an increased size of the acyl pocket (Phe295Ala) had a small effect (~1.5 fold) on the reactivity for the R stereoisomer of non-charged O-ethyl S-3-(isopropyl-4-methylpentyl) methylphosphonothioate (P[R] ncVX) which has the ethoxy moiety towards the acyl pocket [30] and ii) the lack of stereoselectivity for MEPQ (7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide), which has a methyl-and an ethoxy substituent [35]. In the present study, inhibitions of wild type hAChE, Phe295Ala and Phe297Ala by separated fenamiphos enantiomers show that none of the two mutants reverses the stereochemical preference.…”

Organophosphorus compounds as chemical warfare agents: a review

“…53 The residues Trp-84, Glu-199 and Phe-330 are the main components of the anionic subsite (also known as choline binding site), strongly interacting with the quaternary ammonium group of ACh. 45,[61][62][63] Particularly, Trp-84 is very close (3.7 Å) to the ammonium group, and both Trp-84 and Phe-330 interact with this group by cation-π interactions. 64 Although used for long time, the expression "anionic subsite" is not precise, since it contains, at most, one formal negative charge; the cation-π interactions are responsible for the binding of choline to the active site, and not any eventual ionic interaction.…”