人類3-巰基丙酮酸硫轉移酶其脫硫機制之 理論探討

Abstract

人類3-巰基丙酮酸硫轉移酶(human 3-mercaptopyruvate sulfurtransferase, h-MST)能夠緩解人體內的氰中毒，解毒過程包括過硫鍵的生成以及後續的脫硫反應(dethiolation)。在過硫鍵生成的步驟中，3-巰基丙酮酸(3-mercaptopyruvate, 3-MP)上的硫原子轉移到h-MST上的半胱胺酸248殘基(Cys248)上，形成過硫化的Cys248與丙酮酸(pyruvate)。本研究著重探討脫硫反應，首先建構小尺度的CG模型來進行模擬。CG模型是由半胱胺酸─甘胺酸雙肽再加上氰離子(CN-)所構成，並使用密度泛函理論(density functional theory, DFT)及Møller-Plesset 二階微擾理論(the second order Møller-Plesset perturbation theory, MP2)進行結構最佳化及建立反應位能面；而氰離子在這個模型中與過硫化的Cys248反應，經過兩步驟的途徑生成較無毒性的硫氰根離子(SCN-)：分別為第一步驟的過硫鍵斷裂，以及第二步驟的質子轉移。在B3LYP/6-311++G(d,p)計算下得到的第一及第二活化能，分別是55.5 kcal/mol及40.1 kcal/mol；而在MP2的計算下則是55.0 kcal/mol及45.7 kcal/mol。此外，在真實蛋白質系統(PDB code: 4JGT)中則使用兩層的ONIOM計算法進行探討，模擬結果顯示，反應中僅有一個過渡態，脫硫機制為一步驟反應。在ONIOM(B3LYP:AMBER)下計算得到的活化能為45.6 kcal/mol，而在ONIOM(MP2:AMBER)下得到的活化能，則提高至58.4 kcal/mol。綜上所述，我們認為在CG模型與蛋白質系統計算得到的高活化能，暗示了雖然人體中存在h-MST可以幫助解毒，但發生氰中毒時仍會致死的原因。

The human mercaptopyruvate sulfurtransferase (h-MST) is able to alleviate the physiological cyanide poisoning, and the detoxification process involves the formation of persulfide bond followed by dethiolation. At the stage of persulfide formation, the sulfur atom transfers from the 3-mercaptopyruvate (3-MP) to the Cys248 residue of the h-MST and produces the persulfidated Cys248 residue and pyruvate. In this study we aim at the mechanisms of the dethiolation process, and firstly construct the CG model to perform the simulation. The CG model that consists of cysteine-glycine dipeptide and cyanide (CN-) is preliminarily constructed to optimize the geometry, and to depict the potential energy surfaces (PES) using density functional theories (DFT) as well as the second order Møller-Plesset perturbation theory (MP2). In this model, cyanide reacts with the aforementioned persulfidated Cys248 residue and finally produces the less toxic thiocyanate (SCN-) via a two-step mechanism: the first step is the persulfide bond cleavage, while the second step involves the proton transfer. The energy barriers of the two steps are 55.5 kcal/mol and 40.1 kcal/mol at the level of B3LYP/6-311++G(d,p), while MP2 theory gives higher values at 55.0 kcal/mol and 45.7 kcal/mol using triple-zeta 6-311++G(d,p) basis set. In addition, the reaction is further investigated in the protein system (PDB code: 4JGT) utilizing a two-layer ONIOM scheme, and the simulations suggest a concerted route with only one transition state. At ONIOM(B3LYP:AMBER) level, the activation energy is computed at 45.6 kcal/mol, and the activation barrier is elevated to 58.4 kcal/mol at ONIOM(MP2:AMBER) level. In conclusion, the high activation barrier in both of the CG model and the protein system might explain the fatality of cyanide poisoning though the existence of MSTs in vivo.