The hydrolysis of RNase H: Insight into the molecular mechanism via high level 1st principle QM simulation

在我们的研究中，我们将完成我们目前的调查水解的磷酸二酯键之间的两个核苷酸的RNA链的人RNase H，一种酶，结合RNA/DNA杂交双链底物。在我们以前的研究过程中，我们已经确定了这种水解的可能途径，然而，对于酶促反应来说，能量势垒可能太高。就在最近，我们编制了一个非常有前途的替代方案，它与我们最初的假设不同，将反应简化为两步机制。为了详细研究该反应途径，我们将继续使用基于DFT的综合QM/MM理论方法和成熟的轻推弹性弯曲（NEB）计算来指定固定点并确定反应坐标的自由能分布。从突变的酶-底物复合物的X射线结构产生的野生型蛋白质的RNase H结构开始，我们将使用一系列约束和松弛步骤来模拟产物和中间状态。随后将在整个反应坐标上进行过渡态搜索和能垒计算，并确定反应的限速步骤。

In our study we will complete our present investigation on the hydrolysis of the phosphodiester linkages between two nucleotides of RNA chains by human RNase H, an enzyme that binds RNA/DNA hybrid duplex substrates. In the course of our previous studies we have already identified a possible pathway for this hydrolysis, however, with energy barriers presumably too high for an enzymatic reac-tion. Just recently, we have now compiled a very promising alternative, which differs from our original hypothesis by simplifying the reaction to a 2 step mechanism. To investigate this reaction pathway in detail we will continue using a DFT based comprehensive QM/MM theoretical method and the well established nudged elastic bend (NEB) calculations to designate the stationary points and to deter-mine the free energy profile of the reaction coordinates. Starting from the RNase H structure of the wild type protein generated from the X-ray structure of the mutated enzyme-substrate complex we are going to use a series of constrained and relaxation steps to model the product and intermediate states. This will be followed by performing transition state searches and energy barrier calculations over the entire reaction coordinates and the identification of the rate limiting step of the reaction.