Crystal Structures of Acetylcholinesterase in Complex with Organophosphorus Compounds Suggest that the Acyl Pocket Modulates the Aging Reaction by Precluding the Formation of the Trigonal Bipyramidal Transition State^,

Abstract

Organophosphorus compounds (OPs), such as nerve agents and a group of insecticides, irreversibly inhibit the enzyme acetylcholinesterase (AChE) by a rapid phosphorylation of the catalytic Ser203 residue. The formed AChE-OP conjugate subsequently undergoes an elimination reaction, termed aging, that results in an enzyme completely resistant to oxime-mediated reactivation by medical antidotes. In this study, we present crystal structures of the non-aged and aged complexes between Mus musculus AChE (mAChE) and the nerve agents sarin, VX, and diisopropyl fluorophosphate (DFP) and the OP-based insecticides methamidophos (MeP) and fenamiphos (FeP). Non-aged conjugates of MeP, sarin, and FeP and aged conjugates of MeP, sarin, and VX are very similar to the noninhibited apo conformation of AChE. A minor structural change in the side chain of His447 is observed in the non-aged conjugate of VX. In contrast, an extensive rearrangement of the acyl loop region (residues 287-299) is observed in the non-aged structure of DFP and in the aged structures of DFP and FeP. In the case of FeP, the relatively large substituents of the phosphorus atom are reorganized during aging, providing a structural support of an aging reaction that proceeds through a nucleophilic attack on the phosphorus atom. The FeP aging rate constant is 14 times lower than the corresponding constant for the structurally related OP insecticide MeP, suggesting that tight steric constraints of the acyl pocket loop preclude the formation of a trigonal bipyramidal intermediate.

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

“…An adaptable central linker is consistent with the previous suggestion that Ortho-7 can adjust to different conformations of the active-site gorge [7]. Moreover, the acyl loop has previously been described as flexible and various reversible and irreversible ligands are known to induce a structural change in this region [29,[37][38][39]. For example, side-chain rotations of Phe295 and Phe297, similar to the movement observed in Ortho-7fep-mAChE, have been described in the bis-tacrine-bound Torpedo californica AChE complex (pdb code: 2cmf; [39]).…”

“…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].…”

“…Analogously, nonaged and aged forms of diisopropyl fluorophosphate (DFP) and the aged form of fenamiphos induce structural changes of the acyl loop upon conjugating to Ser203 [29,37]. 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.…”

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6

“…In contrast to the structure presented here, the side chain of Glu202 did not interact directly with the sarin moiety; instead, it was coordinated to a water molecule that acted as a bridge between Glu202 and the sarin O2 atom (6,10,19,20). To further investigate the sarin adduct's conformation, The calculated interaction energies for selected fragments in gas phase calculated using the BLYP-D3/ aug-cc-pVTZ method.…”

“…In these structures, the O-isopropyl moiety of sarin is directed toward the indole ring of Trp86 and interacts with Glu202 through a bridging interaction with a conserved water molecule (19). This pose is the preferred conformation of the sarin adduct in the crystal structures of both the human and mouse enzymes (Fig.…”