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Mechanism of oxime reactivation of phosphonylated acetylcholinesterase analyzed by chirality and mutagenesis

Organophosphates inactivate acetylcholinesterase by reacting covalently with the active center serine. We have examined the reactivation of a series of resolved enantiomeric methylphosphonate conjugates of acetylcholinesterase by two oximes, 2-pralidoxime (2-PAM) and 1-(2'-hydroxy iminomethyl-1'-pyridinium)-3-(4' carbamoyl-1-pyridinium) (HI-6). The Sp-enantiomers of the methylphosphonate esters are far more reactive in forming the conjugate with the enzyme, and we find that rates of oxime reactivation also show an Sp versus Rp preference suggesting that a similar orientation of the phosphonyl oxygen towards the oxyanion hole is required for both efficient inactivation and reactivation. A comparison of reactivation rates of Sp- and Rp-cycloheptyl-, 3,3 dimethylbutyl- and isopropyl methylphosphonyl conjugates shows that steric hindrance by the alkoxy group precludes facile access of the oxime to the tetrahedral phosphorus. To facilitate access, we substituted smaller side chains in the acyl pocket of the active center and find that the Phe295Leu substitution enhances the HI-6 elicited reactivation rates of the Sp conjugates up to 14-fold, whereas the Phe297Ile substitution preferentially enhances 2-PAM reactivation by as much as 125-fold. The fractional enhancement of reactivation achieved by these mutations of the acyl pocket is greatest for the conjugated phosphonates of the largest steric bulk. By contrast, little enhancement of reactivation rate is seen with these mutants for the Rp conjugates, where limitations on oxime access to the phosphonate and suboptimal positioning of the phosphonyl oxygen in the oxyanion hole may both slow reactivation. To facilitate access of oximes to RP conjugates from the side of the enzyme choline binding site we substituted Ala for Tyr at position 337. This substitution significantly enhanced reactivation rates of all RP conjugates and two out of three SP conjugates, by both 2PAM and HI-6. Maximal rates of reactivation were primarily increased reflecting an improved geometry of the pentacoordinate transition state with the oxime moiety attacking from choline binding site. Combined substitutions of acyl pocket and choline binding site residues at positions 295 and 337, 297 and 337, and ternary residue substitution at positions 295,297,337 further enhanced reactivation rates only in a few cases. The majority of combined mutations, however, reactivated at similar rates as wildtype acetylcholinesterase or slower, thus approaching reactivation rates of wildtype butyrylcholinesterase. The active center gorge of butyrylcholinesterase is devoid of six aromatic residues, found in acetylcholinesterase, three of which are at positions homologous to 295,297 and 337. Taken together, the above findings suggest that impaction of the conjugated organophosphate within the constraints of the active center gorge is a major factor in influencing oxime access and reactivation rates. While opening of the gorge can greatly enhance oxime efficacy, a gorge devoid of critical aromatic residues as seen in butyrylcholinesterase yields an environment not conductive to efficient reactivation. Moreover, the individual oximes differ in attacking orientation leading to the presumed pentavalent transition state. Hence, efficacies of oximes as reactivating agents are dependent not only on oxime structure, but also the steric bulk of the intervening groups surrounding the tetrahedral phosphorus and the attacking oxime molecule. An efficient cholinesterase-oxime binary combination would convert plasma cholinesterase from a stoichiometric to a catalytic scavenger of organophosphate nerve agents. The catalytic turnover and prophylactic efficacy of the combination will depend on the reactivation rate achieved by the oxime. This work was supported by U.S. Army Medical Research and Materiel Command under Grant project Order DAMD17-18014

Organophosphates; acetylcholinesterase; pyridinium oximes; chirality

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