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

RNase H 的水解：通过高水平第一原理 QM 模拟深入了解分子机制

• 批准号: 52814317
• 负责人: Professor Dr. Gregor Fels
• 金额: --
• 依托单位: Fakultät für Naturwissenschaften
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/52814317?language=en
• 关键词: hydrolysis; RNase; Insight; into; molecular

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.

在我们的研究中，我们将完成我们目前的研究，即人RNaseH对RNA链上两个核苷酸之间的磷酸二酯键的水解，这是一种结合RNA/DNA杂化双链底物的酶。在我们以前的研究过程中，我们已经确定了一种可能的水解途径，但是，假设能量垒太高，不适合进行酶反应。就在最近，我们编制了一个非常有希望的替代方案，它通过将反应简化为两步机制而不同于我们最初的假设。为了详细研究这一反应途径，我们将继续使用基于DFT的综合QM/MM理论方法和成熟的微动弹性弯曲(NEB)计算来指定驻点并确定反应坐标的自由能分布。从突变的酶-底物复合体的X射线结构产生的野生型蛋白质的RNaseH结构出发，我们将使用一系列约束和松弛步骤来模拟产物和中间态。随后将在整个反应坐标上执行过渡态搜索和能垒计算，并识别反应的速率限制步骤。