Hassan Hammad Ali, Rani Sadaf, Fatima Tabeer, Kiani Farooq Ahmad, Fischer Stefan
Research Center for Modeling and Simulation (RCMS), National University of Sciences and Technology (NUST), 44000 Islamabad, Pakistan.
Research Center for Modeling and Simulation (RCMS), National University of Sciences and Technology (NUST), 44000 Islamabad, Pakistan; Department of Biotechnology, University of Gujrat Sialkot Sub Campus, 51310 Sialkot, Pakistan.
Biophys Chem. 2017 Nov;230:27-35. doi: 10.1016/j.bpc.2017.08.003. Epub 2017 Aug 15.
Hydrolysis of phosphate groups is a crucial reaction in living cells. It involves the breaking of two strong bonds, i.e. the OH bond of the attacking water molecule, and the PO bond of the substrate (O and O stand for attacking and leaving oxygen atoms). Mechanism of the hydrolysis reaction can proceed either by a concurrent or a sequential mechanism. In the concurrent mechanism, the breaking of OH and PO bonds occurs simultaneously, whereas in the sequential mechanism, the OH and PO bonds break at different stages of the reaction. To understand how protonation affects the mechanism of hydrolysis of phosphate monoester, we have studied the mechanism of hydrolysis of protonated and deprotonated phosphate monoester at M06-2X/6-311+G**//M06-2X/6-31+G*+ZPE level of theory (where ZPE stands for zero point energy). Our calculations show that in both protonated and deprotonated cases, the breaking of the water OH bond occurs before the breaking of the PO bond. Because the two events are not separated by a stable intermediate, the mechanism can be categorized as semi-concurrent. The overall energy barrier is 41kcalmol in the unprotonated case. Most (5/6th) of this is due to the initial breaking of the water OH bond. This component is lowered from 34 to 25kcalmol by adding one proton to the phosphate. The rest of the overall energy barrier comes from the subsequent breaking of the PO bond and is not sensitive to protonation. This is consistent with previous findings about the effect of triphosphate protonation on the hydrolysis, where the equivalent protonation (on the γ-phosphate) was seen to lower the barrier of breaking the water OH bond and to have little effect on the PO bond breaking. Hydrolysis pathways of phosphate monoester with initial breaking of the PO bond could not be found here. This is because the leaving group in phosphate monoester cannot be protonated, unlike in triphosphate hydrolysis, where protonation of the β- and γ-phosphates had been shown to promote a mechanism where the PO bond breaks before the OH bond does. We also point out that the charge shift due to PO bond breaking during sequential ATP hydrolysis in bio-molecular motors onsets the week unbinding of hydrolysis product that finally leads to the product release during power stroke.
磷酸基团的水解是活细胞中的关键反应。它涉及两个强键的断裂，即进攻水分子的OH键和底物的PO键（O和O分别代表进攻氧原子和离去氧原子）。水解反应的机制可以通过协同或连续机制进行。在协同机制中，OH键和PO键同时断裂，而在连续机制中，OH键和PO键在反应的不同阶段断裂。为了理解质子化如何影响磷酸单酯的水解机制，我们在M06 - 2X/6 - 311 + G**//M06 - 2X/6 - 31 + G* + ZPE理论水平（其中ZPE代表零点能）下研究了质子化和去质子化磷酸单酯的水解机制。我们的计算表明，在质子化和去质子化情况下，水分子的OH键断裂都发生在PO键断裂之前。由于这两个事件之间没有被一个稳定的中间体隔开，该机制可归类为半协同机制。在未质子化的情况下，总能量垒为41 kcal/mol。其中大部分（六分之五）是由于水分子OH键的初始断裂。通过向磷酸基团添加一个质子，这一成分从34 kcal/mol降至25 kcal/mol。总能量垒的其余部分来自随后PO键的断裂，并且对质子化不敏感。这与先前关于三磷酸质子化对水解影响的研究结果一致，在该研究中，等效质子化（在γ - 磷酸上）被发现降低了水分子OH键断裂的能垒，而对PO键断裂影响很小。在这里没有找到磷酸单酯以PO键初始断裂的水解途径。这是因为与三磷酸水解不同，磷酸单酯中的离去基团不能被质子化，在三磷酸水解中，β - 和γ - 磷酸的质子化已被证明促进了一种PO键在OH键之前断裂的机制。我们还指出，在生物分子马达中连续ATP水解过程中，由于PO键断裂引起的电荷转移引发了水解产物的弱解离，最终导致在动力冲程中产物释放。
