Development and application of QM/MM methods for metalloenzymes

项目摘要：金属酶扮演着各种重要的生物角色，因此是主要的目标 用于生物医学研究。它们还推动了计算方法的进一步发展，这些方法可以 在精确度和采样效率之间取得适当的平衡。受到上一笔资金取得进展的鼓舞 期间，我们继续发展量子力学/分子力学(QM/MM)的混合方法来解决这些问题。 了解在关键生物过程中发挥主要作用的金属酶的催化机制，如 磷酸转移和DNA复制。我们将广泛比较动力学同位素效应(KIE)。 和组合突变效应的实验，以校准我们的方法。fic的具体目标是： 1.进一步发展了生物中过渡金属离子的近似密度泛函方法(DFTB3) 申请。这涉及：(I)。改进对金属-配体极化和电荷转移的描述 荷电配体的相互作用，由自然成键轨道分析指导；打造高质量 使用密度矩阵等高度相关的QM方法对金属-配体相互作用的基准数据集 具有正则变换理论的重整化群；(Iii).包括显式的现场d-d交互 在配体-fi场理论框架下的轨道能级而不是布居能级。2.提高对机制的认识-- 金属离子在磷酸转移酶中的作用。通过QM/MM自由能的组合 而KIE的计算，我们将：(一)。解释为什么磷酸酶-1中的磷酰化转移过渡态，但是 不在碱性磷酸酶中，尽管它们的双金属大体相似，但基本上相对于溶液改变了fi 活性中心；确定差异是否由金属离子(锌与锰)的同一性决定， 它们之间的距离或活性部位中电荷/偶极子的分布；(Ii)对催化作用进行量化- 现代时间分辨结晶学研究中发现的⌘聚合酶fi的第三个“瞬变”MG2的归属 研究并建立了该离子对3‘-羟基活化机理的影响。3.集成DFTB3/MM 和DFT/MM方法，以提供对与各种 碱性磷酸酶中关键基序的组合突变识别碱性磷酸酶“催化模块”fi 地点。这些突变体的催化活性范围很广，以kcat/km为单位跨越十个数量级， 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，我们的努力将 帮助建立“最佳实践”的QM/MM方案，以帮助合理设计金属酶和 了解它们的进化。

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.