The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and soman-inhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.
Serving a critical role in neurotransmission, human acetylcholinesterase (hAChE) is the target of organophosphate nerve agents. Hence, there is an active interest in studying the mechanism of inhibition and recovery of enzymatic activity, which could lead to better countermeasures against nerve agents. As hAChE is found in different oligomeric assemblies, certain approaches to studying it have been problematic. Herein, we examine the biochemical and structural impact of monomerizing hAChE by using two mutations: L380R/F535K. The activities of monomeric hAChE L380R/F535K and dimeric hAChE were determined to be comparable utilizing a modified Ellman's assay. To investigate the influence of subunit-subunit interactions on the structure of hAChE, a 2.1 A X-ray crystallographic structure was determined. Apart from minor shifts along the dimer interface, the overall structure of the hAChE L380R/F535K mutant is similar to that of dimeric hAChE. To probe whether the plasticity of the active site was overtly impacted by monomerizing hAChE, the kinetic constants of (PR/S ) - VX (ethyl({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinate) inhibition and subsequent rescue of hAChE L380R/F535K activity with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)) were determined and found to be comparable to those of dimeric hAChE. Thus, hAChE L380R/F535K could be used as a substitute for dimeric hAChE when experimentally probing the ability of the hAChE active site to accommodate future nerve agent threats or judge the ability of new therapeutics to access the active site.
The wild-type OPAA enzyme has relatively high levels of catalytic activity against several organophosphate G-type nerve agents. A series of mutants containing replacement amino acids at the OPAA Y212, V342, and I215 sites showed several fold enhanced catalytic efficiency on sarin, soman, and GP. One mutant, Y212F/V342L, showed enhanced stereospecificity on sarin and that enzyme along with a phosphotriesterase mutant, GWT, which had the opposite stereospecificity, were used to generate enriched preparations of each sarin enantiomer. Inhibition of acetylcholinesterase by the respective enantioenriched sarin solutions subsequently provided identification of the sarin enantiomers as separated by normal phase enantioselective liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry.
