Mechanism of Oxime Reactivation of Acetylcholinesterase Analyzed by Chirality and Mutagenesis

Abstract

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'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium) (HI-6). The S(p) 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 S(p) versus R(p) preference, suggesting that a similar orientation of the phosphonyl oxygen toward the oxyanion hole is required for both efficient inactivation and reactivation. A comparison of reactivation rates of (S(p))- and (R(p))-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 S(p) 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 the reactivation rate is seen with these mutants for the R(p) conjugates, where limitations on oxime access to the phosphonate and suboptimal positioning of the phosphonyl oxygen in the oxyanion hole may both slow reactivation. These 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. Moreover, the individual oximes differ in attacking orientation, leading to the presumed pentavalent transition state. Hence, their efficacies as reactivating agents depend on the steric bulk of the intervening groups surrounding the tetrahedral phosphorus.

Molecular aspects of the reactivation process of acetylcholinesterase inhibited by cyclosarin

“…[2][3][4][5][6] Several molecular modelling studies available in literature point out to important features on the oximes structures that could be very useful to guide experimental research on this issue. [7][8][9][10][11][12][13][14][15][16][17][18][19] In a former work 4 …”

Organophosphorus compounds as chemical warfare agents: a review

“…In general, the phosphylenzymes derived from nerve agents with S P configuration (which are more toxic) are more prone to reactivation; this indicates that polarization of the phosphoryl bond is necessary for an efficient reactivation by nucleophiles. 118,150 Ideally, the oximate and the enzyme (the leaving group in the reactivation) would occupy the axial positions in the trigonal bipyramidal intermediate; however, steric constraints in the active site gorge prevent the optimal attack of the oximate. Because of this, reactivations by oximes are much slower than phosphylation reactions.…”

“…Because of this, reactivations by oximes are much slower than phosphylation reactions. 118,150 The possibility of AChE reinhibition by the phosphylated oxime may pose a serious clinical problem. 151,152 Accordingly, an efficient reactivator should have an unstable phosphylated form.…”

“…143 Structural models for the mechanism of action and the differences in activity among oximes have been proposed. 122,147,150,155,156 It is known that an adequate orientation of the phosphoryl bond inside the active site is necessary for both the enzyme efficient inhibition and its reactivation; however, the orientation of the oximes in the active site and their angles for attacking the phosphylated serine are different. 150 Many new oximes have been synthesized, and their capacities of AChE reactivation evaluated.…”

“…122,147,150,155,156 It is known that an adequate orientation of the phosphoryl bond inside the active site is necessary for both the enzyme efficient inhibition and its reactivation; however, the orientation of the oximes in the active site and their angles for attacking the phosphylated serine are different. 150 Many new oximes have been synthesized, and their capacities of AChE reactivation evaluated. [157][158][159][160][161][162][163][164][165][166][167][168][169][170] Figure 15 shows the structure of some of those new promising bis-pyridinium oximes.…”