Crystal Structures of “Aged” Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase

Organophosphates (OP) are potent transition state (TS) inhibitors which react rapidly with acetylcholinesterase (AChE), and then may undergo an internal dealkylation to produce an irreversibly inhibited, “aged” OP-enzyme conjugate. To understand the structural basis for the stability of aged enzyme, we crystallized and solved the X-ray structures of conjugates obtained by reaction of Torpedo californica (Tc) AChE with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman). After reaction with OP, unbound inhibitor was removed by gel filtration and aging was allowed to proceed to >90% completion. Aged OP-TcAChE was crystallized using PEG-200 and MES buffer at pH 5.8. X-ray data were collected using trigonal crystals, and refined using difference Fourier techniques at 2.2A (DFP), 2.5A (sarin), and 2.2A (soman) resolution. In each structure, the highest positive difference density peak, corresponding to the OP, was observed to be within covalent bonding distance of Ser200. All three structures suggest that the stability of aged AChE derives from interaction of the two resonance oxygen atoms attached to the phosphorus atom with catalytic subsites of the enzyme. Based upon the geometry of the refined structures, we infer that backbone amides of the oxyanion hole (Gly118, Gly119 and Ala201) stabilize one oxygen by hydrogen bonding, while the His440 imidazolium holds the other oxygen in a salt bridge. The conformations of the active sites of aged sarin- and soman-TcAChE are essentially identical and provide structural models for the rate-limiting deacylation TS that occurs during enzyme hydrolysis of the natural substrate, acetylcholine.