Masson P, Froment M T, Bartels C F, Lockridge O
Centre de Recherches du Service de Santé des Armées, Unité de Biochimie, BP 87, 38702 La Tronche Cédex, France and Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA.
Biochem J. 1997 Jul 1;325 ( Pt 1)(Pt 1):53-61. doi: 10.1042/bj3250053.
Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BuChE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in Torpedo AChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3- and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BuChE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BuChE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BuChE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BuChE, allowing some regeneration by spontaneous hydrolysis.
天冬氨酸-70（Asp-70）是人类丁酰胆碱酯酶（BuChE）外周阴离子位点的决定性氨基酸，而乙酰胆碱酯酶还有其他几种氨基酸，其中最重要的是色氨酸-277（电鳐乙酰胆碱酯酶中为色氨酸-279）。我们研究了D70G、D70K和A277W突变体，以评估Asp-70和色氨酸-277在与有机磷酸酯反应中的作用。我们发现，Asp-70对于结合带正电荷的依可硫磷很重要，但对中性对氧磷和异-OMPA不重要。Asp-70对于结合带正电荷的解磷定（2-PAM）以及通过过量2-PAM激活再活化也很重要。过量2-PAM具有类似于底物激活的作用，表明2摩尔2-PAM可结合到野生型BuChE上，而不能结合到D70G突变体上。一个令人惊讶的结果是，Asp-70对于不可逆老化很重要，D70G突变体对氧磷抑制的和二异丙基氟磷酸抑制的BuChE的老化速率分别低3倍和8倍。Asp-70突变体的磷酸化速率常数和2-PAM再活化速率常数与野生型相同。A277W突变体在所有测定中表现得与野生型一样。我们的结果预测，具有非典型（D70G）变体BuChE的人对依可硫磷的毒性作用将更敏感，但对氧磷和二异丙基氟磷酸将同样敏感。具有D70G突变的人其被抑制的BuChE将对2-PAM的再活化有抗性，但这将被被抑制的BuChE较低的不可逆老化速率所抵消，从而允许通过自发水解进行一些再生。
