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The presence of a precisely aligned active-site triad (Ser-His-Asp/Glu) in the three-dimensional structures of widely different hydrolytic enzymes has generated intense interest in the chemical modus operandi of this catalytic motif. 1 One hypothesis, which has not received wide acceptance, proposes that the imidazole of the catalytic His is mobile during enzyme function. 2 We solved the structures of the phosphonylation and dealkylation ("aging") reaction products of acetylcholinesterase (AChE; EC 3.1.1.7) and an organophosphorus (OP) inhibitor, O-ethyl-S-[2-[bis(1-methylethyl)amino]ethyl] methylphosphonothioate (VX) by X-ray crystallography. The structures clearly demonstrate reversible movement of the catalytic His. Moreover, the conformational change apparently involves a hydrogen (H-) bond with a glutamate (E199) which had been implicated previously in OP and substrate reactions.Most serine hydrolases, including AChE, use a catalytic triad and a dipolar oxyanion hole in tandem to catalyze substrate hydrolysis via an acylation-deacylation mechanism. 3 This twostep mechanism is also a weakness, however, because it renders the enzyme susceptible to stoichiometric inhibition by "hemisubstrates" which react to form stable analogues of natural reaction intermediates. Following phosphonylation of the active-site Ser Oγ, some OP-enzyme adducts undergo further post-inhibitory reactions, including dealkylation, which result in truly irreversible enzyme inhibition (collectively called "aging") (Scheme 1).Structures of the reaction products of Torpedo californica (Tc) AChE with DFP, sarin or soman 4 after aging reveal that the OP undergoes dealkylation to yield a stable anionic phosphonyl adduct. 5 As reported previously for aged OP-serine proteases, 6 the catalytic imidazole (H440) of TcAChE is positioned to form H-bonds with its normal carboxylic acid partner (E327), and with one oxygen of the negatively charged phosphonyl moiety. Such structures are limited, however, because they reveal only the final product (II) of the OP reaction. To overcome this limitation, we employed VX. Although phosphonylation of AChE with VX is rapid (>10 7 M -1 min -1 ), the ethyl group of VX dealkylates slowly, thus allowing us to solve the structures of both (I) and (II) by conventional X-ray crystallography to 2.2 and 2.4 Å resolution, respectively. 7 The most striking feature of the pro-aged VX-AChE structure (I) was disruption of the catalytic triad due to movement of H440. The H440 N∂ was 4.5 Å away from its resting state partner, E327

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