Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

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• 作者：L.G. Sultatos ; R. Kaushik
• 关键词：Organophosphorus insecticides ; Organophosphates ; Acetylcholinesterase ; Peripheral anionic site
• 刊名：Toxicology and Applied Pharmacology
• 出版年：2008
• 出版时间：1 August 2008
• 年：2008
• 卷：230
• 期：3
• 页码：390-396
• 全文大小：648 K

文摘

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 B_max and K_d for 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 B_max did 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 B_max, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K_d represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K_d, and dimethylphosphorylation of Ser203 decreasing K_d. 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.