Mutation Report for: E202Q_mouse-ACHE

To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationic m-trimethylammoniotrifluoroacetophenone (TFK+) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE. Hence it appears that TFK+ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of +4.

It has been demonstrated that cholinesterases (ChEs) are an effective mode of pretreatment to prevent organophosphate (OP) toxicity in mice and rhesus monkeys. The efficacy of ChE as a bioscavenger of OP can be enhanced by combining enzyme pretreatment with oxime reactivation, since the scavenging capacity extends beyond a stoichiometric ratio of ChE to OP. Aging has proven to be a major barrier to achieving oxime reactivation of acetylcholinesterase (AChE) inhibited by the more potent OPs. To further increase the stoichiometry of OP to ChE required, we have sought AChE mutants that are more easily reactivated than wild-type enzyme. Substitution of glutamine for glutamate (E199) located at the amino-terminal to the active-site serine (S200) in Torpedo AChE generated an enzyme largely resistant to aging. Here we report the effect of the corresponding mutation on the rate of inhibition, reactivation by 1-(2-hydroxyiminomethyl-1-pyridinium)-1(4-carboxyaminopyridinium)- dimethyl ether hydrochloride (HI-6), and aging of mouse AChE inhibited by C(+)P(-)- and C(-)P(-)-epimers of soman. The E202 to Q mutation decreased the affinity of soman for AChE, slowed the reactivation of soman-inhibited AChE by HI-6, and decreased the aging of mutant AChE. These effects were more pronounced with C(-)P(-)-soman than with C(+)P(-)-soman. In vitro detoxification of soman and sarin by wild-type and E202Q AChE in the presence of 2 mM HI-6 showed that, E202Q AChE was 2-3 times more effective in detoxifying soman and sarin than wild-type AChE. These studies show that these recombinant DNA-derived AChEs are a great improvement over wild-type AChE as bioscavengers. They can be used to develop effective methods for the safe disposal of stored OP nerve agents and potential candidates for pre- or post-exposure treatment for OP toxicity.