Structure-Function Studies of Ribonucleotide Reductase

核糖核苷酸还原酶的结构-功能研究

• 批准号: 10387984
• 负责人: DANIEL G. NOCERA
• 金额: $ 8.23万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10387984
• 关键词: Active Sites; Address; Affinity; Amides; Amino Acids; Anabolism; Anti-Bacterial Agents; Anti-Inflammatory Agents; Anti-Retroviral Agents; Bacterial Antibiotic Resistance; Biochemical; Biological; Biological Assay; Biological Process; Biology; Chemicals; Chemistry; Clinical; Complex; Coupled; Coupling; Cryoelectron Microscopy; Cysteine; DNA; DNA biosynthesis; Deoxyribonucleotides; Development; Disease; Drug Design; Drug Targeting; Electron Transport; Electrons; Enzymatic Biochemistry; Enzymes; Event; Experimental Designs; Face; Future; Generations; Health; Heart; Human; Individual; Kinetics; Lasers; Life; Link; Malignant Neoplasms; Mediating; Methods; Modality; Modeling; Molecular Biology; Molecular Conformation; Nature; Nucleotides; Outcome; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Pharmaceutical Preparations; Phase; Play; Process; Protons; Reaction; Regulation; Resolution; Ribonucleotide Reductase; Ribonucleotides; Role; Site; Specificity; Spectrum Analysis; Stretching; Structure; Sulfhydryl Compounds; System; Techniques; Therapeutic Uses; Tryptophan; Variant; Visualization; Work; base; cancer therapy; chemotherapy; cofactor; design; holistic approach; inhibitor/antagonist; innovation; insight; instrumentation; interdisciplinary approach; novel therapeutics; nucleic acid metabolism; protein structure; repaired; small molecule therapeutics; therapeutic target; unnatural amino acids; vibration

Abstract Proton-coupled electron transfer (PCET) is a ubiquitous mechanism in biology, serving as the basis for mediating steps involving biosynthesis of metabolites, radical generation and transport, and the activation of substrates at cofactors. The control of highly reactive radical intermediates is achieved by coupling proton and electron transfer processes. Management of radicals in biology is of particular relevance to human health, as enzymes operating by PCET are therapeutic targets with wide-ranging applications including chemotherapy, anti-retroviral and anti-bacterial drugs and anti-inflammatory agents. Of the enzymes that operate by PCET, ribonucleotide reductases (RNRs) are exceptional in their biological function and are paramount to health, as the enzymes produce the DNA building blocks for life. The central role of RNRs in nucleic acid metabolism has made the human RNR the target of five clinically used therapeutics that shut down the PCET pathway and, consequently, nucleotide reduction. The class Ia RNR is the exemplar of biological PCET; its function originates from a reversible long-range radical transport pathway that spans 35 Å and two subunits (α and β) upon every turnover. An interdisciplinary approach integrates biochemical methods with the transient spectroscopy afforded by the requested instrumentation to target three specific aims. Specific Aim 1 seeks to address the role of PCET in nucleotide reduction, both in the substrate activation phase involving the conserved radical at the “top face” of the active site, as well as in the radical substrate reduction phase at the “bottom face” of the active site. Work will be advanced by interfacing the TRIR instrumentation with existing laser instrumentation to define kinetics of key intermediates associated with individual steps of RNR active site chemistry and with model compounds that faithfully capture the RNR active site chemistry. The TRIR technique will also be used the follow amide I and II stretches of key residues in amino acid networks that govern allosteric PCET regulation between the α and β subunits. This work is guided by new structural insights afforded from cryo-EM studies, which allow both the nature of subunit interactions and the networks of amino acids that connect the catalytic, specificity, and activity sites of the intact enzyme to be identified. The structural and temporal visualization of subunit dynamics that come from these studies will inform on the design of new small molecule therapeutics targeting the subunit interface. The TRIR will also aid in studies of Specific Aim 3 that utilize biochemical and molecular biology innovations to elucidate initial events of radical transfer within the β-subunit, with a focus on a critical tryptophan within the PCET pathway.

抽象的 质子耦合电子传输（PCET）是生物学中普遍存在的机制，它是 中介步骤涉及代谢产物的生物合成，激进的产生和运输以及激活 辅助因子的底物。通过将质子耦合和 电子传输过程。生物学辐射的管理与人类健康特别相关，因为 由PCET运行的酶是具有大范围应用的热靶标，包括化学疗法， 抗逆转录病毒和抗菌药物和抗炎药。由PCET运行的酶的 核糖核苷酸还原酶（RNR）在其生物学功能方面是例外，对健康至关重要，如 这些酶生产了DNA构建块。 RNR在核酸代谢中的核心作用具有 使人类RNR成为五种临床使用疗法的靶标，该疗法关闭了PCET途径，并且 因此，核苷酸还原。 IA RNR类是生物PCET的典范。它的功能 起源于可逆的远程自由基运输途径，该途径跨越35Å和两个亚基（α和β） 在每个失误上。跨学科方法将生化方法与瞬态相结合 所请求的仪器为针对三个特定目的而提供的光谱法。特定目标1试图 解决了PCET在核苷酸还原中的作用，这两者在底物激活阶段都涉及 在活性位点的“顶面”以及在自由基底物还原阶段的保守自由基 活动站点的“底部”。将通过将Trir仪器与现有 激光仪器定义与RNR活动位点各个步骤相关的关键中间体的动力学 化学和模型化合物，可完美捕获RNR活性位点化学。 trir 技术还将使用以下酰胺I和II延伸的氨基酸网络中的关键保留率 控制α和β亚基之间的变构PCET调节。这项工作以新的结构见解为指导 来自冷冻EM研究提供的，既允许亚基相互作用的性质和氨基网络 连接要鉴定的完整酶的催化，特异性和活性位点的酸。这 这些研究所产生的亚基动态的结构和临时可视化将告知 针对亚基界面的新小分子疗法的设计。 TRIR还将有助于研究 利用生化和分子生物学创新来阐明自由基的初始事件的特定目标3 在β-亚基内转移，重点是PCET途径内的关键色氨酸。