The Relationship of Protein dynamics to Enzyme Catalysis

蛋白质动力学与酶催化的关系

• 批准号: 6382613
• 负责人: NORBERT O. REICH
• 金额: $ 14.04万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6382613
• 关键词: X ray crystallography; chemical kinetics; conformation; enzyme activity; enzyme mechanism; enzyme structure; methionine; methyltransferase; molecular dynamics; mutant; protein structure function; structural biology

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.

描述(由申请人提供)：首席调查员及其 合作者建议研究蛋白质动力学在 提高酶促甲基转移的催化速率。在备受关注的同时 在酶催化领域，过渡状态是稳定的 或者是对支持蛋白质重要性的证据--熵的争论 对催化的动力学研究很少。蛋白质肯定会经历各种不同的 构象变化的类型；例如，支持相关的证据 有助于配体结合的运动是可用的。相似关联运动 已经被鉴定为几种酶催化的反应，使用分子 动态模拟。研究人员建议使用x射线的组合。 结晶学、MD模拟和功能分析，以提供 蛋白质动力学与酶相关的实验和理论基础 反应速度。他们建议研究一种名为S-腺苷的细菌Hhai 蛋氨酸依赖的DNA胞嘧啶C5甲基转移酶。S-腺苷 蛋氨酸依赖的酶在生物学中广泛存在，并能修饰细胞核 酸、蛋白质、脂类、碳水化合物以及药物。哺乳动物和 细菌DNA甲基转移酶是抗癌和抗肿瘤的重要药物靶点 分别是抗生素类药物。海先生是最被理解的S-腺苷 任何类型的蛋氨酸依赖甲基转移酶，因为许多 高分辨率的共晶结构是可用的，动力学和 化学机制是众所周知的。首席调查员和他的 合作者确定了看似重要的结构元素 通过检查M.HhaI-DNA共晶体结构来研究蛋白质动力学 对同一结构进行分子动力学分析。已经准备了几个突变体，并 正在进行结晶学、多重构象(MCA)和MD分析。 他们建议将这种结构和动态信息与 甲基转移速率的测量。数据分析将重点放在 甲基化与1)蛋白质柔韧性、2)原子间的相关性 距离，3)相关运动，4)近攻击构象的频率 (NAC)，以及5)如果是异构体的分布。分子的多重构象分析 WT M.Hhai-DNA和突变的共晶结构将用于设计更多 以测试甲基化和相关运动之间的关系。一个 基于提取的筛查将遵循涉及到的片段的随机突变 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 应该提供独特的见解，具有潜在的广泛影响， 相关的构象变化有助于催化作用。M.Hhai代表一种 一类具有重要医疗应用的酶，以及更好的 机械属性。