Zhang Xin, Wu Ruibo, Song Lingchun, Lin Yuchun, Lin Menghai, Cao Zexing, Wu Wei, Mo Yirong
Department of Chemistry, Western Michigan University, Kalamazoo, Michigan 49008, USA.
J Comput Chem. 2009 Nov 30;30(15):2388-401. doi: 10.1002/jcc.21238.
Combined QM(PM3)/MM molecular dynamics simulations together with QM(DFT)/MM optimizations for key configurations have been performed to elucidate the enzymatic catalysis mechanism on the detoxification of paraoxon by phosphotriesterase (PTE). In the simulations, the PM3 parameters for the phosphorous atom were reoptimized. The equilibrated configuration of the enzyme/substrate complex showed that paraoxon can strongly bind to the more solvent-exposed metal ion Zn(beta), but the free energy profile along the binding path demonstrated that the binding is thermodynamically unfavorable. This explains why the crystal structures of PTE with substrate analogues often exhibit long distances between the phosphoral oxygen and Zn(beta). The subsequent SN2 reaction plays the key role in the whole process, but controversies exist over the identity of the nucleophilic species, which could be either a hydroxide ion terminally coordinated to Zn(alpha) or the micro-hydroxo bridge between the alpha- and beta-metals. Our simulations supported the latter and showed that the rate-limiting step is the distortion of the bound paraoxon to approach the bridging hydroxide. After this preparation step, the bridging hydroxide ion attacks the phosphorous center and replaces the diethyl phosphate with a low barrier. Thus, a plausible way to engineer PTE with enhanced catalytic activity is to stabilize the deformed paraoxon. Conformational analyses indicate that Trp131 is the closest residue to the phosphoryl oxygen, and mutations to Arg or Gln or even Lys, which can shorten the hydrogen bond distance with the phosphoryl oxygen, could potentially lead to a mutant with enhanced activity for the detoxification of organophosphates.
结合量子力学（PM3）/分子力学分子动力学模拟以及对关键构型的量子力学（密度泛函理论）/分子力学优化，以阐明磷酸三酯酶（PTE）对对氧磷解毒的酶催化机制。在模拟中，对磷原子的PM3参数进行了重新优化。酶/底物复合物的平衡构型表明，对氧磷可以强烈结合到溶剂暴露程度更高的金属离子Zn(β)上，但沿结合路径的自由能分布表明这种结合在热力学上是不利的。这解释了为什么PTE与底物类似物的晶体结构中磷氧与Zn(β)之间的距离往往很长。随后的SN2反应在整个过程中起关键作用，但对于亲核物种的身份存在争议，亲核物种可能是末端与Zn(α)配位的氢氧根离子，也可能是α-和β-金属之间的微羟基桥。我们的模拟支持后者，并表明限速步骤是结合的对氧磷发生扭曲以接近桥连羟基。在这个准备步骤之后，桥连氢氧根离子攻击磷中心并以低势垒取代二乙基磷酸酯。因此，设计具有增强催化活性的PTE的一种合理方法是稳定变形的对氧磷。构象分析表明，Trp131是最接近磷酰氧的残基，将其突变为Arg或Gln甚至Lys，可以缩短与磷酰氧的氢键距离，这可能会产生一种对有机磷酸酯解毒活性增强的突变体。
