Structure-Function Studies of Ribonucleotide Reductase

核糖核苷酸还原酶的结构-功能研究

基本信息

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
项目类别：
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

项目摘要

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转移（PCET）是生物学中普遍存在的机制，是介导步骤涉及代谢物的生物合成、自由基的产生和运输以及自由基的激活高反应性自由基中间体的控制是通过质子和质子的偶联来实现的。生物学中自由基的管理与人类健康特别相关，因为 PCET 操作的酶是具有广泛应用的治疗靶点，包括化疗、 PCET 发挥作用的抗逆转录病毒药物、抗菌药物和抗炎药物，核糖核苷酸还原酶 (RNR) 具有特殊的生物学功能，对健康至关重要，因为 RNR 在核酸代谢中发挥着核心作用。使人类 RNR 成为五种临床使用的治疗方法的目标，这些治疗方法可关闭 PCET 途径，并且，因此，Ia 类 RNR 是生物 PCET 功能的典范；源自跨越 35 Å 和两个亚基（α 和 β）的可逆长程自由基传输途径每次营业额都采用跨学科方法将生化方法与瞬态相结合。特定目标 1 旨在通过所需仪器提供的光谱来实现三个特定目标。解决 PCET 在核苷酸还原中的作用，两者都在涉及活性位点“顶面”的保守自由基，以及自由基底物还原相活动站点的“底面”工作将通过将 TRIR 仪器与现有的接口来推进。激光仪器定义与 RNR 活性位点各个步骤相关的关键中间体的动力学化学和忠实捕获 RNR 活性位点化学的模型化合物。技术还将用于氨基酸网络中关键残基的以下酰胺 I 和 II 段，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。冷冻电镜研究提供了亚基相互作用的性质和氨基网络连接待鉴定的完整酶的催化、特异性和活性位点的酸。来自这些研究的亚基动力学的结构和时间可视化将告知针对亚基界面的新小分子疗法的设计也将有助于研究。具体目标 3 利用生化和分子生物学创新来阐明激进的初始事件 β-亚基内的转移，重点是 PCET 途径中的关键色氨酸。