Aging of Cholinesterases Phosphylated by Tabun Proceeds through O-Dealkylation

Abstract

Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.

Crystal structures of oxime-bound fenamiphos-acetylcholinesterases: Reactivation involving flipping of the His447 ring to form a reactive Glu334–His447–oxime triad

“…The acyl loop of the previously reported nonaged form of fep-mAChE adopts a conformation found in the apo mAChE structure; however, due to the low occupancy of the phosphorous conjugate in fep-mAChE, the reported stereochemistry as well as the position of the acyl loop [29] need further confirmation. Despite the apo-like conformation acyl loop in HI-6•fep-mAChE, it is likely that aging of fenamiphos occur via the same mechanism as described for tabun, i.e., His447 is involved as a catalyst for aging and subsequently stabilizes the negatively charged dealkylated product [40].…”

HLö-7 - A REVIEW OF ACETYLCHOLINESTERASE REACTIVATOR AGAINST ORGANOPHOSPHOROUS INTOXICATION

“…soman 2-4 minutes; sarin 5-12 hours; tabun 46 hours; VX 48 hours) which is important for better antidote treatment. [16][17][18][19] The similarity between murine AChE (mAChE) and hAChE helped to describe in detail the mechanism of OPs binding. 20 The OPs binding is directed by its covalent bond with the catalytic serine (Ser203 on hAChE).…”

Organophosphorus compounds as chemical warfare agents: a review

“…In the case of the nerve agent tabun, aging occurs by breaking of a P-N bond, instead of a PO-C bond, with elimination of a dimethylamine group, 90,91 however, the possibility of occurring a classical aging for tabun has been recently proven. 92 93 by loss of two isopropyl groups in the complex with HuAChE 94 (as represented in Figure 7), and by loss of just one isopropyl group in the complex with equine BChE. 95 The aged phosphylated cholinesterase cannot be reactivated due to several factors.…”