The pH Dependence of Dealkylation in Soman-Inhibited Cholinesterases and Their Mutants:  Further Evidence for a Push−Pull Mechanism

Abstract

Bimolecular rate constants for the inactivation of recombinant (r) human (Hu) butyrylcholinesterase (BChE) with P(S)C(S)- and P(S)C(R)-2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) are (92 +/- 7) x 10(6) M-1 min-1 and (13.7 +/- 0.8) x 10(6) M-1 min-1 at pH 7.4, mu = 0.1 M and 25 degreesC. Mutations of E197(199) to D or Q and W82(84) to A result in reductions in the rate constants for inactivation with P(S)C(S)-soman 4.3-, 11.8-, and 263-fold and with P(S)C(R)-soman by 6.5-, 47.3-, and 685-fold, respectively. The pH dependence of dealkylation (aging) in r mouse (Mo) acetylcholinesterase (AChE) and rHu BChE and their mutants inactivated with P(S)C(S)- and P(S)C(R)-soman was compared. Best-fit parameters for the asymmetric bell curves for the adducts of wild-type Mo AChE are pK1 = pK2 = 4.0-4.9 and pK3 = 5.2-6.6. These pKs are consistent with the involvement of two carboxylic acids, possibly E202(199) and either E334(327) or E450(443), and H447(440)H+ in the dealkylation of AChE. E202Q MoAChE inactivated with the soman diastereomers yielded pK3 = 5.5-5.8. Nearly symmetric pH curves for soman-inhibited wild-type and E197D Hu BChE gave pK2 = 3.7-4.6 and pK3 = 7.3-8.0, but much lower, pK3 approximately 5, for the corresponding adduct of the E197Q mutant. Dealkylation in soman-inhibited BChE is consistent with the participation of one carboxylic acid side chain and H438(440)H+. Maximal rate constants for dealkylation (kmax) are 1-6 min-1 for AChE and 2 min-1 for BChE at 25 degreesC. The W82 to A mutation in BChE results in the largest reduction, 2500-6000-fold, in the rate constant for dealkylation. The reduction in the rate constants for dealkylation in the E197 mutants is highly pH dependent. The solvent isotope effects at the pH maxima are 1.3-1.4, indicating unlikely preprotonation or proton in "flight" at the enzymic transition states. The new results support the push-pull mechanism of dealkylation in soman-inhibited cholinesterases proposed previously.

Molecular dynamics study of active-site interactions with tetracoordinate transients in acetylcholinesterase and its mutants

“…The basic tenet of our model for the mechanism of dealkylation in soman-inhibited ChEs is the electrostatic ' push-pull ' mechanism of transition-state stabilization in Scheme 1 [6,[12][13][14][15]. Several experimental facts support this model.…”

“…(4) The upper pK calculated from the pH rate profile for dealkylation is consistent with that of histidine H + -440. (5) The Glu199Gln mutant of T. californica AChE [16], Glu202Gln mutant of mouse AChE [14], Glu202Gln mutant of human AChE [17,18], Trp86Ala and Phe330Ala mutants of human AChE [19,20] and the corresponding mutations in human butyrylcholinesterase [14,15,21] showed much diminished capacity for dealkylation when inhibited with soman. (6) 99 % of the product of the reaction is derived from a tertiary rather than a secondary carbenium ion [22].…”

“…Dealkylation in the P S diastereomers of 2-(3,3-dimethylbutyl)-methylphosphonofluoridate (soman)-inhibited ChEs occurs at approx. 10 orders of magnitude faster than in a non-enzymic model reaction [12][13][14][15] ; the question is why ?…”

Organophosphorus compounds as chemical warfare agents: a review

“…96 Finally, at least in the case of the nerve agent soman, it has been proposed the existence of a "push-pull" electrostatic mechanism, involving residues Glu-199, His-440, Trp-84 and the oxyanion hole in TcAChE, which would stabilize the carbenium formed during aging and favor a methyl migration that would transform a secondary carbenium in a tertiary one. 97,99,103,104 Spontaneous reactivation rates of phosphylated cholinesterases are determined by the structure of the phosphyl-enzyme. Except for the nerve agents, spontaneous reactivation occurs at clinically significant rates with the majority of the OPCs, but it is always slower than the deacetylation that happens in the hydrolysis of the natural substrate: Deacetylation of AChE occurs in microseconds, but dephosphylation in hours to days.…”

“…It is believed that the rapid aging of soman is due to the formation of a tertiary carbenium (which later rearranges itself in neutral compounds) by a methyl migration in the secondary pinacolyl carbenium aided by a 'push-pull' mechanism, as shown in Figure 10. 97,99,103,104 Table 4 compiles data of rate constants for inhibition of HuAChE by racemic mixtures of several nerve agents, and also presents rate constants for aging and spontaneous reactivation of the phosphylated enzyme in each case.…”