Mangas I, Taylor P, Vilanova E, Estévez J, França T C C, Komives E, Radić Z
Unit of Toxicology and Chemical Safety, Institute of Bioengineering, University Miguel Hernandez of Elche, Alicante, Spain.
Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, 92093, USA.
Arch Toxicol. 2016 Mar;90(3):603-16. doi: 10.1007/s00204-015-1481-1. Epub 2015 Mar 6.
The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.
丝氨酸水解酶乙酰胆碱酯酶（AChE）活性中心催化三联体丝氨酸的羟基氧原子会在磷原子处攻击有机磷化合物（OPs），取代一级离去基团并形成共价键。被抑制的AChE可以通过自发地或与亲核试剂（如肟）反应切断丝氨酸 - 磷键而重新激活。同时，被抑制的AChE加合物会在一个称为老化的过程中随着时间的推移通过逐步脱烷基作用失去部分分子。老化酶的重新激活尚未得到证实。在此，我们的目标是研究被丙氟磷或沙林类似物（Flu - MPs，荧光甲基膦酸酯）抑制的人AChE的肟重新激活和老化反应。使用肟2 - PAM抑制Flu - MPs后观察到逐步重新激活。然而，用2 - PAM或更有效的肟抑制丙氟磷后未观察到重新激活。通过基质辅助激光解吸电离飞行时间质谱（MALDI - TOF）和基质辅助激光解吸电离串联飞行时间质谱（MALDI - TOF/TOF）开发了一种肽指纹质谱（MS）方法，该方法能够清晰地区分与OPs加合的人AChE的带有活性丝氨酸的肽（活性中心肽，ACP）。在对氧磷抑制后检测到带有二乙基磷酸化加合物的ACP，在Flu - MPs抑制并随后老化后检测到带有异丙基甲基膦酰化和甲基膦酰化加合物的ACP。然而，在丙氟磷抑制并用肟孵育后观察到未老化的未重新激活的复合物，其中MS数据显示带有NN - 二异丙基磷酰加合物的ACP。动力学实验表明活性没有重新激活。对丙氟磷抑制的人AChE进行的计算分子模型分析，绘制了脱烷基作用分离的原子之间的能量与距离的关系图，结果显示能量需求很高，因此老化概率很小。然而，对于Flu - MPs和二异丙基氟磷酸酯（DFP），在我们的MS数据和先前发表的晶体结构中观察到了老化现象，建模计算的能量需求较低，因此老化似乎是更可能发生的反应。尽管我们没有观察到老化现象，但我们在此记录了磷酸化人AChE对肟重新激活具有抗性的直接证据。
