Carletti Eugénie, Li He, Li Bin, Ekström Fredrik, Nicolet Yvain, Loiodice Mélanie, Gillon Emilie, Froment Marie T, Lockridge Oksana, Schopfer Lawrence M, Masson Patrick, Nachon Florian
Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, 38700 La Tronche, France.
J Am Chem Soc. 2008 Nov 26;130(47):16011-20. doi: 10.1021/ja804941z.
Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.
人丁酰胆碱酯酶（hBChE）可水解或清除多种有毒酯类，包括海洛因、可卡因、氨基甲酸酯类农药、有机磷农药和神经毒剂。有机磷酸酯（OPs）通过催化丝氨酸的磷酸化抑制乙酰胆碱酯酶（AChE），从而发挥其急性毒性。磷酸化胆碱酯酶（ChE）可经历一个自发的、时间依赖性的过程，称为“老化”，在此过程中，OP-ChE共轭物会发生脱烷基化。这会导致酶的不可逆抑制。塔崩对ChEs的抑制作用以及随后的老化反应尤为引人关注，因为塔崩-ChE共轭物对目前大多数肟类复活剂表现出极强的抗性。我们通过测定被塔崩抑制的hBChE未老化和老化形式的晶体结构，并更新被塔崩抑制的未老化和老化小鼠AChE（mAChE）的精修结果，研究了磷酰胺化ChE共轭物肟抗性的结构基础。未老化和老化的塔崩-hBChE结构分别精修至2.3 Å和2.1 Å。老化ChE共轭物的精修结构清楚地表明，老化反应是通过塔崩P（R）对映体的O-脱烷基化进行的。脱烷基化后，带负电荷的氧与质子化的His438Nε2形成强盐桥，从而阻止复活。对老化的塔崩抑制的hBChE进行质谱分析表明，磷上的二甲胺和乙氧基侧链均缺失。乙氧基的缺失与晶体学结果一致。二甲胺的缺失与质谱分析中老化加合物制备过程中的酸催化脱酰胺作用一致。所报道的三维数据将有助于设计能够复活塔崩-ChE共轭物的新型肟类。
