Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun^,

Abstract

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 Å resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acylenzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10 4 -fold more lethal P S stereoisomer of soman relative to the P R form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to deadend organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.The organophosphorus (OP) nerve agents sarin, soman, tabun, and VX are among the deadliest man-made chemicals (1). While the military use of OP nerve agents is widely banned, these compounds have been employed in recent decades by rogue states and terrorist groups. In 1988, Iraq employed weaponized sarin against its own Kurdish citizens in Halabja, a town adjacent to the Iranian border, killing an estimated 5,000 (2). Coordinated attacks on the Tokyo subway system by the Japanese Aum Shinrikyo cult in 1995 also employed sarin, killing 12 and injuring † This work was supported, in part, by NIH grants CA98468 and NS58089, and the American Lebanese Syrian Associated Charities. ‡ Protein Data Bank codes are 2HRQ for the hCE1-soman structure and 2HRR for the hCE1-tabun structure. *Corresponding Author: Matthew R. Redinbo, Ph.D., Department of Chemistry, CB #3290, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, (919)843-8910; Fax: (919)962-2388, redinbo@unc.edu. 1 Abbreviations: AcChE, Human acetylcholinesterase; BuChE, Human butyrylcholinesterase; CE, carboxylesterase; hCE1, Human carboxylesterase 1; HI-6, 1-(2-hydroxy-iminomethylpyridinium)-1-(4-carboxyamino)-pyridinium dimethylether dichloride; MuAcChE, Mus musculus acetylcholinesterase; OP, organophosphate; Ortho-7, 1,7-heptylene-bis-N,N′-2-pyridiniumaldoxime dichloride; PON1, human serum paraoxonase 1; rmsd, root mean square deviation; Sarin, methylethyl methylphosphonofluoridate; Soman, Tabun, ethyl N, TcAcChE, Torpedo californica acetylcholinesterase; VX, OP toxicity is thought to be mediated through the inhibition of human acety...

Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance

“…S5, no evidence of aging was observed (27). This finding is consistent with previous work that has shown mammalian CBEs to be resistant to the aging reaction (28,29).…”

“…First, activation and stabilization of the alkyl side chain is thought to involve the catalytic histidine, which is positioned close to the alkyl side chain and stabilized in a position to allow this interaction by Glu199 and Phe331 (Torpedo californica AChE numbering). In comparison, the catalytic histidine (His471) in LcαE7 is farther away from the alkyl side chain (3.2 vs. 2.5 Å) and not well-oriented to interact with this group during dealkylation; the catalytic histidine in human CBE is also poorly positioned to interact with the alkyl side chain of a phosphoylated serine (28). Although Glu217 is present in a similar position to Glu199 in AChE, there is no residue present in LcαE7 in the same position as Phe331 in AChE, meaning that the catalytic histidine cannot be stabilized in the required position to catalyze dealkylation.…”

Human Carboxylesterase 1 Stereoselectively Binds the Nerve Agent Cyclosarin and Spontaneously Hydrolyzes the Nerve Agent Sarin

“…In this report, we present X-ray crystallographic and biochemical data designed to evaluate the ability of hCE1 to act as potential enzyme-based therapeutic for nerve agent detoxification. Although we have previously determined crystal structures of hCE1 in complexes with soman and tabun (Fleming et al, 2007), here we report the novel crystal structure of hCE1 inhibited by cyclosarin (Fig. 2), one of the most potent inhibitors of human AChE (Gray and Dawson, 1987).…”

“…hCE1 Stereoselectivity for Nerve Agents 513 lipases, and carboxylesterases (Fleming et al, 2007;Li et al, 2008). As such, these toxins are among the deadliest compounds developed by humans.…”

“…hCE1 cleaves ester, thioester, and amine-ester bonds in a variety of endogenous and xenobiotic compounds (Redinbo and Potter, 2005). We published previously the crystal structures of hCE1 in covalent acyl-enzyme complexes with the nerve agents soman and tabun (Fleming et al, 2007). However, the ability of hCE1 to reverse covalent inhibition and reactivate wild-type activity was not examined in that study.…”