Luo C, Saxena A, Smith M, Garcia G, Radić Z, Taylor P, Doctor B P
Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA.
Biochemistry. 1999 Aug 3;38(31):9937-47. doi: 10.1021/bi9905720.
Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LüH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LüH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LüH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LüH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.
重新激活有机磷酸酯（OP）抑制的乙酰胆碱酯酶（AChE）是治疗OP中毒的关键目标。本研究使用天然、野生型和突变体重组DNA表达的AChE，每种均被代表性的OP化合物抑制，建立了依酚氯铵加速肟诱导的OP-AChE缀合物再激活与吡啶鎓肟LüH6和TMB4再激活过程中再激活酶的磷酰肟抑制之间的关系。由于该肟形成的磷酰肟极其不稳定，使用HI-6作为再激活剂时未观察到这种反复抑制。首次分离出LüH6和TMB4使乙氧基甲基膦酰基-AChE缀合物再激活过程中形成的磷酰肟，并通过（31）P NMR确认其结构。然而，二乙基磷酰基-AChE缀合物再激活过程中形成的磷酰肟稳定性不足以通过（31）P NMR检测到。用LüH6和TMB4使乙氧基甲基膦酰基-AChE缀合物再激活过程中形成的纯化乙氧基甲基膦酰肟作为AChE抑制剂的效力比MEPQ高10至22倍，并且在HEPES缓冲液中pH 7.2下可稳定数小时。在兔血清对氧磷酶存在下，这两种肟对乙氧基甲基膦酰基和二乙基磷酰基-AChE的再激活均加速，表明有机磷水解酶可水解再激活过程中形成的磷酰肟。我们的结果强调，某些肟，如LüH6和TMB4，如果用于治疗OP农药中毒，可能由于磷酰肟的形成而导致AChE的长期抑制。
