Ridder Lars, Rietjens Ivonne M C M, Vervoort Jacques, Mulholland Adrian J
School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom.
J Am Chem Soc. 2002 Aug 21;124(33):9926-36. doi: 10.1021/ja0256360.
Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide substrates. In the present study, combined quantum mechanical/molecular mechanical (QM/MM) simulations of the conjugation of glutathione to phenanthrene 9,10-oxide, catalyzed by the M1-1 isoenzyme from rat, have been carried out to obtain insight into details of the reaction mechanism and the role of solvent present in the highly solvent accessible active site. Reaction-specific AM1 parameters for sulfur have been developed to obtain an accurate modeling of the reaction, and QM/MM solvent interactions in the model have been calibrated. Free energy profiles for the formation of two diastereomeric products were obtained from molecular dynamics simulations of the enzyme, using umbrella sampling and weighted histogram analysis techniques. The barriers (20 kcal/mol) are in good agreement with the overall experimental rate constant and with the formation of equal amounts of the two diastereomeric products, as experimentally observed. Along the reaction pathway, desolvation of the thiolate sulfur of glutathione is observed, in agreement with solvent isotope experiments, as well as increased solvation of the epoxide oxygen of phenanthrene 9,10-oxide, illustrating an important stabilizing role for active site solvent molecules. Important active site interactions have been identified and analyzed. The catalytic effect of Tyr115 through a direct hydrogen bond with the epoxide oxygen of the substrate, which was proposed on the basis of the crystal structure of the (9S,10S) product complex, is supported by the simulations. The indirect interaction through a mediating water molecule, observed in the crystal structure of the (9R,10R) product complex, cannot be confirmed to play a role in the conjugation step. A selection of mutations is modeled. The Asn8Asp mutation, representing one of the differences between the M1-1 and M2-2 isoenzymes, is identified as a possible factor contributing to the difference in the ratio of product formation by these two isoenzymes. The QM/MM reaction pathway simulations provide new and detailed insight into the reaction mechanism of this important class of detoxifying enzymes and illustrate the potential of QM/MM modeling to complement experimental data on enzyme reaction mechanisms.
谷胱甘肽S-转移酶（GSTs）在哺乳动物对外源化学物质的解毒过程中发挥着重要作用。它们催化谷胱甘肽与多种亲电化合物的结合。菲9,10-氧化物是GSTs的一种模型底物，代表了一类重要的环氧化物底物。在本研究中，对大鼠M1-1同工酶催化谷胱甘肽与菲9,10-氧化物结合的过程进行了量子力学/分子力学（QM/MM）联合模拟，以深入了解反应机制的细节以及高溶剂可及活性位点中溶剂的作用。已开发出硫的反应特异性AM1参数以获得反应的精确建模，并对模型中的QM/MM溶剂相互作用进行了校准。使用伞形采样和加权直方图分析技术，从酶的分子动力学模拟中获得了两种非对映体产物形成的自由能分布。屏障（20千卡/摩尔）与整体实验速率常数以及两种非对映体产物等量形成的情况非常吻合，这与实验观察结果一致。沿着反应途径，观察到谷胱甘肽硫醇盐硫的去溶剂化，这与溶剂同位素实验一致，同时菲9,10-氧化物的环氧化物氧的溶剂化增加，说明了活性位点溶剂分子的重要稳定作用。已识别并分析了重要的活性位点相互作用。基于（9S,10S）产物复合物的晶体结构提出的Tyr115通过与底物环氧化物氧直接形成氢键的催化作用得到了模拟的支持。在（9R,10R）产物复合物的晶体结构中观察到的通过介导水分子的间接相互作用，无法证实在结合步骤中起作用。对一系列突变进行了建模。Asn8Asp突变代表了M1-1和M2-2同工酶之间的差异之一，被确定为可能导致这两种同工酶产物形成比例差异的一个因素。QM/MM反应途径模拟为这类重要的解毒酶的反应机制提供了新的详细见解，并说明了QM/MM建模在补充酶反应机制实验数据方面的潜力。
