Unique Push−Pull Mechanism of Dealkylation in Soman-Inhibited Cholinesterases

Abstract

The pH-dependence and solvent isotope effects of dealkylation in diastereomeric adducts of Electric eel (Ee) and fetal bovine serum (FBS) acetylcholinesterase (AChE) inactivated with P(-)C(+) and P(-)C(-) 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) were studied at 4.0 +/- 0.2 degrees C. The rate constant versus pH profiles were fit to a bell-shaped curve for all adducts. Best fit parameters are pK1 4.4-4.6 and pK2 6.3-6.5 for Ee AChE and pK1 4.8-5. 0 and pK2 5.8 for FBS AChE. The pKs are consistent with catalytic participation of the Glu199 anion and HisH+440. Maximal rate constants (kmax) are 13-16 x 10(-3) s-1 for Ee AChE and 8 x 10(-3) s-1 for FBS AChE. The solvent isotope effects at the pH maxima are 1.1-1.3, indicating unlikely proton transfer at the enzymic transition states for the dealkylation reaction. Slopes of log rate constant versus pH plots are near 1 at 25.0 +/- 0.2 degrees C between pH 7.0 and 10.0. In stark contrast, the corresponding adducts of trypsin are very stable even at 37.0 +/- 0.2 degrees C. The rate constants for diastereomers of soman-inhibited trypsin at 37.0 +/- 0.2 degrees C are pH independent and approximately 10(4) times smaller than kmax for analogous adducts with AChE. Dealkylation in soman-inhibited AChEs is estimated to occur at >10(10) times faster than a plausible nonenzymic reaction. Up to 40% of the catalytic acceleration can be attributed to an electrostatic push, and an electrostatic pull provides much of the balance. The results of this work together with results of a product analysis by Michel et al. (1969) can be explained by an initial and rate-determining methyl migration from Cbeta to Calpha. This is driven by the high electron density of residues (Glu199 and Trp84) at a crowded active site and may be concerted with C-O bond breaking. The positive charge at the rate-determining transition state is distributed between Cbeta and His440. A tertiary carbocation may have a fleeting existence before it is trapped by water or neighboring electrons which is likely to be promoted by Glu199 as the proton acceptor.

The effect of neutral oximes on the reactivation of human acetylcholinesterase inhibited with paraoxon

“…Furane-2-carboxyaldehyde oxime (23) It was prepared using 0.16 mL (2 mmol) of furane-2-carboxyaldehyde. After recrystallization with methanol, 0.173 g (78%) and 0.197 g (89%) of the oxime were obtained as amber crystals with conventional and microwave heating, respectively; mp 71-73 °C (Lit.…”

“…The aging process occurs at different times, depending on the nature of the organophosphorus compound. 1,[21][22][23][24][25] An understanding of the processes and mechanisms behind the reactivation of the inhibited AChE (11) is required to develop new drugs for the treatment of intoxication with neurotoxic organophosphorus agents. 10 The most effective reported reactivation compounds are cationic oximes prepared from pyridine aldehydes.…”

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.…”

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

“…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 ?…”