Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos.
Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos.
复制标题
毒死蜱或敌敌畏磷酸化活性位点后，硫黄素 t 与乙酰胆碱酯酶外周阴离子位点的结合发生改变。
DOI：
10.1016/j.taap.2008.03.006
复制
发表时间：
2008
影响因子：
3.8
通讯作者：
Kaushik,R
中科院分区：
文献类型：
--
作者：
Sultatos,LG;Kaushik,R
乙酰胆碱酯酶的外围阴离子位点，当被配体占据时，已知调节该重要酶的活性位点处的反应速率。目前的报告利用外周阴离子位点特异性荧光探针硫磺素T，以确定是否有机磷毒死蜱oxon和敌敌畏结合到人重组乙酰胆碱酯酶的外周阴离子位点，因为某些有机磷显示浓度依赖性动力学抑制这种酶时。用50 nM或2000 nM抑制剂孵育3 nM乙酰胆碱酯酶活性位点改变了硫磺素t与外周阴离子位点结合的Bmax和Kd。然而，这些变化是由Ser203的磷酸化引起的，因为将任一抑制剂从50 nM增加至2000 nM并不改变进一步的硫磺素t结合动力学。此外，有机磷酸酯诱导的Bmax降低并不代表结合位点的实际减少，而是可能由酰化和外围阴离子位点之间的构象相互作用导致结合的硫磺素t的刚性降低。荧光量子产率的下降，导致Bmax的明显降低，将伴随着结合的硫磺素t分子的刚性降低。有机磷诱导的K的变化代表了硫磺素t的结合亲和力的变化，其中Ser203的二乙基磷酸化增加Kd，Ser203的二甲基磷酸化降低Kd。这些结果表明，毒死蜱氧磷和敌敌畏不直接结合到乙酰胆碱酯酶的外周阴离子位点，但可以通过Ser203的磷酸化影响结合到该位点。
The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the Bmaxand Kdfor thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in Bmaxdid not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in Bmax, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in Kdrepresented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing Kd, and dimethylphosphorylation of Ser203 decreasing Kd. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.