Development and application of QM/MM methods for metalloenzymes

项目概述：金属酶具有多种重要的生物学作用，因此是主要的靶点 用于生物医学研究。它们还推动了计算方法的进一步发展， 准确性和采样效率的适当平衡。对上一次筹资取得的进展感到鼓舞， 在此期间，我们继续发展混合量子力学/分子力学（QM/MM）方法， 了解金属酶的催化机制，在关键的生物过程中发挥重要作用，如 磷酰基转移和DNA复制。我们将对动力学同位素效应（KIE）进行广泛的比较 和组合突变效应与实验来校准我们的方法。具体目标是： 1.进一步发展了一种近似密度泛函方法（DFTB 3），用于生物样品中过渡金属离子的研究。 应用.这包括：（一）。改进了对金属配体极化和电荷转移的描述 带电配体的相互作用，由自然成键轨道分析指导;（ii）。建立高质量 使用高度相关的QM方法（如密度矩阵）的金属-配体相互作用基准数据集 重正化群与正则变换理论;（iii）.包括在现场的直接的D-D互动， 在配位场理论的框架下，轨道而不是布居水平。2.加强机械理解- 研究金属离子在磷酰转移酶中的作用。通过QM/MM自由能的组合 和KIE计算，我们将：（i）。解释为什么磷酸酶-1中的磷酰基转移过渡态，但 不存在于碱性磷酸酶中，相对于溶液显著修饰艾德，尽管它们通常具有相似的酶活性 活性位点;确定差异是否取决于金属离子的身份（Zn 2 + vs. Mn 2+）， 它们之间的距离或活性位点中电荷/偶极子的分布;（ii）.量化催化反应， 在最近的时间分辨晶体学中鉴定的第三种“瞬时”Mg 2+到DNA聚合酶的转化 研究，并建立该离子对3 'OH活化机制的影响。3.集成DFTB 3/MM 和DFT/MM方法，以提供与各种 碱性磷酸酶中的“催化模块”通过活性磷酸酶中关键基序的组合突变而被鉴定为艾德。 绝佳的价钱这些突变体的催化活性范围广泛，kcat/Km跨越十个数量级， 为测试和校准QM/MM方法提供了前所未有的机会。从长远来看，我们的努力 帮助建立“最佳实践”QM/MM协议，能够帮助合理设计金属酶， 了解他们的进化。

Project Summary: Metalloenzymes play various important biological roles and therefore are major targets for biomedical research. They also drive further development of computational methodologies that can strike the proper balance of accuracy and sampling efficiency. Encouraged by progress made in the last funding period, we continue to develop hybrid quantum mechanical/molecular mechanical (QM/MM) methods to un- derstand the catalytic mechanism of metalloenzymes that play major roles in key biological processes such as phosphoryl transfers and DNA replication. We will conduct extensive comparison of kinetic isotope effect (KIE) and combinatorial mutation effects with experiments to calibrate our methodologies. The specific aims are: 1. Further develop an approximate Density Functional method (DFTB3) for transition metal ions in biological applications. This involves: (i). improving the description of polarization and charge transfer of metal-ligand interactions for charged ligands, guided by the Natural Bonding Orbital analysis; (ii). establishing high quality benchmark dataset for metal-ligand interactions using highly correlated QM methods such as Density Matrix Renormalization Group with Canonical Transform theory; (iii). including explicit on-site d - d interactions at the orbital rather than population level in the framework of ligand-field theory. 2. Enhance mechanistic understand- ing in the roles of metal ions in phosphoryl transfer enzymes. Through a combination of QM/MM free energy and KIE calculations, we will: (i). explain why is the phosphoryl transfer transition state in phosphatase-1, but not in alkaline phosphatase, substantially modified relative to solution, despite their generally similar bimetallic active sites; establish whether the difference is dictated by the identity of the metal ions (Zn2+ vs. Mn2+), the distance between them or the distribution of charges/dipoles in the active site; (ii). quantify the catalytic con- tribution of the third “transient” Mg2+ to DNA polymerase ⌘ identified in recent time-resolved crystallography studies, and establish the impact of this ion on the mechanism of 3'OH activation. 3. Integrate DFTB3/MM and DFT/MM methodologies to provide a mechanistic understanding of co-operativity associated with various “catalytic modules” identified in alkaline phosphatase through combinatorial mutation of key motifs in the active site. The broad range of catalytic activities of these mutants, which span ten orders of magnitude in kcat/Km, provides an unprecedented opportunity to test and calibrate QM/MM methods. In the long run, our efforts will help establish “best-practice” QM/MM protocols that are able to aid rational design of metalloenzymes and understand their evolution.