Crosslinking Ribonucleotide Reductase Subunits to Study PCET Within

描述（由申请人提供）：核糖核苷酸还原酶（RNR）负责将核糖核苷酸（NDP）还原为脱氧核糖核苷酸（dNDP），并且在所有生物体中都是必需的，以制备用于DNA复制和修复的单体前体。这项研究计划概述了I类RNR结构的发展，其中两个亚基或一个亚基模拟物共价连接。叠氮基和炔基官能度分别并入R1和R2亚基中，允许它们通过[3+2]环加成进行位点特异性连接。亚基的共价连接将降低被认为参与PCET途径的氨基酸残基的构象灵活性。沿着提出的PCET途径的氨基酸的这种构象限制和系统突变将促进构建体中自由基物质的积累，从而允许通过光谱观察提出的自由基中间体。观察途径中每个残基的自由基种类将证明它们各自参与脱氧核糖核苷酸还原的机制，并允许确定这些PCET事件的速率。这些速率的温度和同位素依赖性的研究将确定这些过程是否受到蛋白质构象变化或PCET过程本身的限制。 成功完成本提案中概述的研究将提供对核糖核苷酸还原酶中自由基运输机制的详细机械理解。公共卫生相关性将哺乳动物细胞中核糖核苷酸减少的机制与细菌和病毒细胞中的核糖核苷酸减少的机制进行比较，将有助于深入了解药物的开发，这些药物可以特异性靶向细菌和病毒RNR中基于自由基的中间体，从而抑制它们的复制和修复，而不会破坏类似的哺乳动物过程。

DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs) are responsible for reducing ribonucleotides (NDPs) to deoxyribonucleotides (dNDPs) and are required in all organisms in order to make the monomeric precursors used in DNA replication and repair. This research proposal outlines the development of class I RNR constructs in which the two subunits or a subunit mimic are covalently attached. Incorporation of azido and alkynyl functionality into to R1 and R2 subunits, respectively, allows for their site-specific attachment via a [3+2] cycloaddition. The covalent attachment of the subunits will reduce the conformational flexibility of the amino acid residues believed to be involved in the PCET pathway. This conformation restriction and systematic mutation of the amino acids along the proposed PCET pathway will facilitate the build up of radical species in the construct thereby allowing the proposed radical intermediates to be observed spectroscopically. Observation of the radical species of each of the residues in the pathway will prove their respective involvement in the mechanism of deoxyribonucleotide reduction and allow for the rates of these PCET events to be determined. Studies of the temperature and isotopic dependence of these rates will determine if these processes are limited by protein conformational changes or the PCET process itself. The successful completion of the research outlined in this proposal will provide a detailed mechanistic understanding of the radical transport mechanism in ribonucleotide reductase. PUBLIC HEALTH RELEVANCE Comparing the mechanism of ribonucleotide reduction in mammalian cells to that in bacterial and viral cells will provide insight into the development of drugs that can specifically target the radical-based intermediates in the bacterial and viral RNR, thereby inhibiting their replication and repair without disrupting the analogous mammalian processes.