Centre de Recherches du Service de Santé des Armées, Dept. Toxicology, 38702 La Tronche Cedex, France.
Chem Biol Interact. 2010 Sep 6;187(1-3):157-62. doi: 10.1016/j.cbi.2010.03.027. Epub 2010 Mar 23.
Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed "aging", results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes. Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge. MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in (18)O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.
磷酸化胆碱酯酶（ChE）可能会发生侧反应，导致其可复性逐渐降低。这个过程称为“老化”，是由于加合物的脱烷基化引起的，取决于有机磷部分的结构。老化的 ChE 对肟的再激活具有抗性。由于有机磷化合物的毒理学重要性，老化的分子机制已经成为研究的课题数十年。目前还不清楚老化是否涉及相同的键断裂，无论 OP 的类型如何，或者是否涉及磷酸/膦酸盐中的 P-O-C 键（P-O 或 O-C）、磷酰胺中的 P-N-C 键和膦硫代酸盐中的 P-S-C 键的断裂。人们假设，老化加合物上磷原子上带负电荷的原子阻止了肟的亲核攻击，但对带负电荷的模型分子的研究并不支持这一假设。老化酶构象灵活性的降低可能会阻止再激活剂在活性位点峡谷中的适当调整，从而导致其不可再激活。水和（18）O 水中磷酸化人丁酰胆碱酯酶（hBChE）的 MALDI-TOF 质谱分析提供了证据，表明老化是由于 O-C 断裂，即 O-脱烷基化引起的。相比之下，异马拉硫磷-BChE 结合物主要通过 P-S 键断裂而老化，但次要产物则来自 O-C 和/或 S-C 断裂。与沙林抑制的 hBChE 和 hAChE 的晶体结构表明，沙林-ChE 结合物的老化是由于 O-脱烷基化引起的。然而，根据 O-烷基和 N-烷基链的性质，其他磷酰胺抑制的 BChE 的老化要么是由于 O-C 断裂，要么是脱氨，即 P-N 断裂。研究发现，支链烷氧基的脱烷基化涉及瞬态碳正离子。OP-ChE 结合物的老化伴随着酶构象变化。人 BChE 的脲、有机溶剂、热和压力变性表明，与天然酶相比，老化的 OP-BChE 结合物的构象稳定性大大增加。不同 OP 结合的 BChE 和 AChE 的三维结构测定表明，老化的加合物与质子化的催化组氨酸形成盐桥。老化酶的结构改变伴随着水分子从酶的活性位点峡谷中逸出。此外，中子散射研究提供了证据表明，与天然酶相比，老化 BChE 的结构动力学发生了显著改变。老化分子基础的知识将有助于设计老化 ChE 的再激活剂、能够减缓老化过程的分子以及假催化 ChE 为基础的生物清除剂。
