The role of AChE active site gorge in determining stereoselectivity of charged and noncharged VX enantiomers

Chem Biol Interact. 2005 Dec 15:157-158:191-8. doi: 10.1016/j.cbi.2005.10.026. Epub 2005 Nov 10.


The reactivity of human acetylcholinesterase (HuAChE) toward the chemical warfare agent VX [O-ethyl S-[2-(diisopropylamino)ethyl] methyl-phosphonothioate] and its stereoselectivity toward the P(S)-enantiomer were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere nc-VX [O-ethyl S-(3-isopropyl-4-methyl-pentyl) methylphosphonothioate]. Stereoselectivity of the wild-type HuAChE toward VX(S) is manifested by a 115-fold higher bimolecular rate constant (1.4 x 10(8) min(-1) M(-1)) as compared to that of VX(R). HuAChE was also 12,500-fold more reactive toward VX(S) than toward nc-VX(S), demonstrating the significance of the polar interactions of the ammonium substituent to their overall affinity toward VX. Indeed, substitution of the cation-binding subsite residue Trp86 by alanine resulted in a decrease of three orders of magnitude in HuAChE reactivity toward both VX enantiomers, with only a marginal effect on the reactivity toward the enantiomers of nc-VX. These results demonstrate that accommodation of the charged moieties of both VX enantiomers depends predominantly on interactions with the aromatic moiety of Trp86. Yet, these interactions seem to limit the stereoselectivity toward the P(S)-enantiomer, which for charged methylphosphonates is much lower than for the noncharged analogs, like sarin or soman. 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 by asparagine (D74N) practically abolished stereoselectivity toward VX(S) (a 130-fold decrease), while substitution which retained 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 originates predominantly from a different orientation of the charged leaving group with respect to residue Asp74. Such different orientations of the charged leaving group in the HuAChE adducts of the VX enantiomers seem to be a consequence of intramolecular interactions with the bulky phosphorus alkoxy group.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholinesterase / chemistry*
  • Acetylcholinesterase / genetics
  • Acetylcholinesterase / metabolism*
  • Acylation
  • Anions / chemistry
  • Binding Sites
  • Choline / chemistry
  • Humans
  • Molecular Structure
  • Mutation / genetics
  • Organothiophosphorus Compounds / chemistry*
  • Organothiophosphorus Compounds / pharmacology*
  • Phenylalanine / genetics
  • Phenylalanine / metabolism
  • Stereoisomerism


  • Anions
  • Organothiophosphorus Compounds
  • Phenylalanine
  • VX
  • Acetylcholinesterase
  • Choline