Arbuzov Institute of Organic and Physical Chemistry, Federal Research Center "Kazan Scientific Center of the Russian Academy of Sciences", Arbuzov str. 8, 420088 Kazan, Russia.
Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin str 4, 119334 Moscow, Russia.
Molecules. 2020 Mar 17;25(6):1371. doi: 10.3390/molecules25061371.
Enzyme-catalyzed hydrolysis of echothiophate, a P-S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of and phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with = 0.20 ± 0.03 mM and = 5.4 ± 1.6 min. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency ( = 2.6 ± 0.2 mM; = 53400 min). With a = (2.6 ± 1.6) × 10 Mmin, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P-S bonded OPs by thiol-free OP hydrolases.
用 Calbiochem Probe IV 作为巯基试剂，通过荧光光谱法监测了 P-S 键有机磷（OP）模型物依可硫磷的酶促水解。所用的 OP 水解酶有：能够水解 OP 的人丁酰胆碱酯酶 G117H 突变体，以及设计为以高速度水解包括 V 类毒剂在内的多种 OP 的磷酸三酯酶 GG1 多重突变体。对 Probe IV 与 OP 水解酶（G117H 丁酰胆碱酯酶、GG1、 和 磷酸三酯酶的野生型以及人对氧磷酶-1）之间的相互作用进行了分子建模。该方法的高灵敏度使得能够对高度纯化的 G117H 丁酰胆碱酯酶浓度低至 0.85 nM 时依可硫磷的水解进行稳态动力学分析。水解呈米氏动力学， = 0.20 ± 0.03 mM， = 5.4 ± 1.6 min。GG1 磷酸三酯酶以高效率水解依可硫磷（ = 2.6 ± 0.2 mM； = 53400 min）。GG1 的 值为（2.6 ± 1.6）×10 Mmin，符合潜在催化生物清除剂的要求条件。量子力学/分子力学（QM/MM）和分子对接表明，Probe IV 与所选的磷酸三酯酶没有显著相互作用。此外，G117H 突变体的结果表明，Probe IV 不会抑制丁酰胆碱酯酶。因此，Probe IV 可推荐用于监测无巯基 OP 水解酶对 P-S 键合 OP 的水解。
