The Relationship of Protein dynamics to Enzyme Catalysis

描述（由申请人提供）：主要研究者及其 合作者建议研究蛋白质动力学对 酶促甲基转移的催化速率增强。虽然很多注意力 在酶催化领域，过渡态稳定化 或熵的论点，证据支持蛋白质的重要性， 催化动力学是缺乏的。众所周知，蛋白质会经历各种 构象变化的类型;例如，支持相关 有助于配体结合的运动是可用的。相似相关运动 已经确定了几种酶催化反应，使用分子 动态模拟研究人员建议将X射线和 晶体学，MD模拟和功能分析，以提供一个 蛋白质动力学与酶相关的实验和理论基础 反应速率他们建议研究M.HhaI，一种细菌S-腺苷 甲硫氨酸依赖的DNA胞嘧啶C5甲基转移酶。S-adenosyl 甲硫氨酸依赖性酶广泛存在于生物学中， 酸、蛋白质、脂质、碳水化合物以及药物。哺乳动物和 细菌DNA甲基转移酶是抗癌的重要药物靶标， 抗生素药物，分别。HhaI是最好理解的S-腺苷 甲硫氨酸依赖的甲基转移酶的任何类型，因为许多 高分辨率共晶结构是可用的， 化学机制是众所周知的。主要研究者及其 合作者确定了结构要素，似乎是重要的， 通过检查M.HhaI-DNA共晶体结构 并对同一结构进行MD分析。已经制备了几种突变体， 正在进行晶体学、多重构象异构体（MCA）和MD分析。 他们建议将这些结构和动态信息与 甲基转移速率的测量。数据分析将集中在 甲基化与1）蛋白质柔性，2）原子间 距离，3）相关运动，4）近攻击构象的频率 （NAC），和5）分布，如果构象。多构象分析 WT M.HhaI-DNA和突变体共晶体结构将用于设计额外的 突变体来测试甲基化和相关运动之间的关系。一个 基于提取物的筛选将遵循涉及以下的片段的随机诱变： 相关运动;候选突变体将提交给结构和 功能分析 结构、分子动力学和功能分析的结合 应该提供具有潜在广泛影响的独特见解， 相关的构象变化有助于催化。HhaI先生代表一个 一类具有重要医学应用的酶，以及一种更好的 机械属性

DESCRIPTION (provided by the applicant): The principal investigator and his collaborators propose to investigate the importance of protein dynamics on the catalytic rate enhancement of enzymatic methyl transfer. While much attention in the field of enzymatic catalysis is given to transition state stabilization or to entropic arguments, evidence in support of the importance of protein dynamics to catalysis is scant. Proteins are certainly known to undergo various types of conformational changes; for example, evidence in support of correlated motions contributing to ligand binding is available. Similar correlated motions have been identified for several enzyme-catalyzed reactions using molecular dynamic simulations. The investigators propose to apply a combination of x-ray crystallography, MD simulations, and functional analyses to provide an experimental and theoretical basis for relating protein dynamics and enzyme reaction rates. They propose to investigate M.HhaI, a bacterial S-adenosyl methionine-dependent DNA cytosine C5 methyltransferase. S-adenosyl methionine-dependent enzymes are widespread in biology and modify nucleic acids, proteins, lipids, carbohydrates, as well as drugs. Mammalian and bacterial DNA methyltransferases are important drug targets for anticancer and antibiotic drugs, respectively. M.HhaI is the best understood S-adenosyl methionine-dependent methyltransferase of any type, since numerous high-resolution cocrystal structures are available and both the kinetic and chemical mechanisms are well known. The principal investigator and his collaborators identified structural elements that appear to be important for protein dynamics through inspection of both the M.HhaI-DNA cocrystal structure and MD analysis of the same structure. Several mutants have been prepared and are undergoing crystallographic, multiple conformer (MCA), and MD analyses. They propose to compare this structural and dynamic information with measurements of the methyl transfer rate. Data analysis will focus on correlations between methylation and 1) protein flexibility, 2) interatomic distances, 3) correlated motions, 4) frequency of near attack conformations (NACs), and 5) distribution if conformers. Multiple conformer analysis of the WT M.HhaI-DNA and mutant cocrystal structures will be used to design additional mutants to test the relationship between methylation and correlated motions. An extract-based screen will follow random mutagenesis of segments implicated in correlated motions; candidate mutants will be submitted to the structural and functional analyses. The combination of structural, molecular dynamic, and functional analyses should provide unique insights with potentially broad impact, into how correlated conformational changes contribute to catalysis. M.HhaI represents a class of enzymes with significant medical applications, and a better mechanistic property.