A.E. Arbuzov Institute of Organic and Physical Chemistry of Russian Academy of Sciences, Arbuzov str. 8, Kazan 420088, Russia.
N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygina str. 4, Moscow 119334, Russia.
Biosci Rep. 2018 Jun 29;38(3). doi: 10.1042/BSR20180609.
The role of water in oxime-mediated reactivation of phosphylated cholinesterases (ChEs) has been asked with recurrence. To investigate oximate water structure changes in this reaction, reactivation of paraoxon-inhibited human acetylcholinesterase (AChE) was performed by the oxime asoxime (HI-6) at different pH in the presence and absence of lyotropic salts: a neutral salt (NaCl), a strong chaotropic salt (LiSCN) and strong kosmotropic salts (ammonium sulphate and phosphate HPO). At the same time, molecular dynamic (MD) simulations of enzyme reactivation under the same conditions were performed over 100 ns. Reactivation kinetics showed that the low concentration of chaotropic salt up to 75 mM increased the percentage of reactivation of diethylphosphorylated AChE whereas kosmotropic salts lead only to a small decrease in reactivation. This indicates that water-breaker salt induces destructuration of water molecules that are electrostricted around oximate ions. Desolvation of oximate favors nucleophilic attack on the phosphorus atom. Effects observed at high salt concentrations (>100 mM) result either from salting-out of the enzyme by kosmotropic salts (phosphate and ammonium sulphate) or denaturing action of chaotropic LiSCN. MDs simulations of diethylphosphorylated hAChE complex with HI-6 over 100 ns were performed in the presence of 100 mM (NH)SO and 50 mM LiSCN. In the presence of LiSCN, it was found that protein and water have a higher mobility, i.e. water is less organized, compared with the ammonium sulphate system. LiSCN favors protein solvation (hydrophobic hydration) and breakage of elelectrostricted water molecules around of oximate ion. As a result, more free water molecules participated to reaction steps accompanying oxime-mediated dephosphorylation.
水在肟介导的磷酰化胆碱酯酶（ChE）重激活中的作用一直受到关注。为了研究该反应中肟的水结构变化，在不同 pH 值下，使用肟类化合物 asoxime（HI-6）对人乙酰胆碱酯酶（AChE）进行了重激活，同时存在和不存在溶致盐：中性盐（NaCl）、强离液盐（LiSCN）和强亲液盐（硫酸铵和磷酸 HPO）。同时，在相同条件下对酶重激活进行了 100 ns 的分子动力学（MD）模拟。重激活动力学表明，低浓度的离液盐（高达 75 mM）增加了二乙基膦酰化 AChE 的重激活百分比，而亲液盐仅导致重激活略有下降。这表明，破水剂盐会破坏围绕肟离子的水分子的去结构化。肟的去溶剂化有利于亲核攻击磷原子。在高盐浓度（>100 mM）下观察到的影响，要么是亲液盐（磷酸和硫酸铵）对酶的盐析作用，要么是离液盐 LiSCN 的变性作用。在存在 100 mM（NH）SO 和 50 mM LiSCN 的情况下，对二乙基膦酰化 hAChE 与 HI-6 复合物进行了 100 ns 的 MDs 模拟。在 LiSCN 的存在下，与硫酸铵体系相比，发现蛋白质和水的流动性更高，即水的组织化程度更低。LiSCN 有利于蛋白质的溶剂化（疏水性水合）和围绕肟离子的电限制水分子的断裂。结果，更多的游离水分子参与了伴随肟介导的去磷酸化的反应步骤。
