Aging of di-isopropyl-phosphorylated human butyrylcholinesterase

Abstract

Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.

Aging-Resistant Organophosphate Bioscavenger Based on Polyethylene Glycol-Conjugated F338A Human Acetylcholinesterase

“…Indeed, here we demonstrate that incorporation of the F338A mutation into PEGylated HuAChE decelerates aging, so that the soman-inhibited enzyme can be reactivated by oximes in vivo to an extent that enables mice to withstand repeated exposure to high doses of soman (e.g., 5.4 LD 50 and then 4 LD 50 ). It should be noted in this context, that attempts to generate a recombinant BChEbased slow-aging enzyme by replacement of residue Phe329, which is analogous to Phe338 in HuAChE, were not successful and had a very limited effect on the rate of aging of BChE (Masson et al, 1997). …”

In vitroreactivation of trichlorfon-inhibited butyrylcholinesterase using HI-6, obidoxime, pralidoxime and K048

“…Efficacy of reactivation strongly decreases (R decrease about 10% for all of reactivators in concentration 1 mM) when time interval of trichlorfon and BChE preincubation is prolonged from half an hour to one hour. These facts respond to expectancies especially when we considered spontaneous dealkylation (aging) of trichlorfon bound in enzyme active centre and no chance to reactive dealkyled organophosphate from this centre [24,25]. The difference between half an hour and one hour preincubation before reactivation application is significant on probability level approx.…”

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

“…(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].…”