MECHANISM AND INHIBITION OF RIBONUCLEOTIDE REDUCTASES

核糖核苷酸还原酶的机制和抑制

• 批准号: 6031066
• 负责人: JOANNE STUBBE
• 金额: $ 5.4万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6031066
• 关键词: DNA replication; Escherichia coli; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; allosteric site; cell growth regulation; chemical models; cobalt; cofactor; dithiol; electron spin resonance spectroscopy; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; free radicals; iron compounds; metalloenzyme; nucleic acid sequence; nucleotide analog; oxidoreductase inhibitor; protein engineering; ribonucleotide reductase; synthetic nucleotide; tyrosine analog; vitamin B12 coenzyme

DESCRIPTION: Ribonucleotide reductases (RNRs) catalyze the rate determining step in DNA biosynthesis conversion of nucleotides to deoxynucleotides. Their central role in nucleic acid metabolism has made them the recent target for design of antitumor and antiviral agents. Their unusual metallo cofactors and their function in initiating the radical dependent nucleotide reduction proces by formation of a thiyl radical has been of interest to inorganic and organic chemists alike. 1. Efforts will focus on understanding the trigger for long range coupled electron/proton transfer, approximately 35 Angstrom, between the two subunits of the class I reductases. Efforts will continue to investigate how the class II RNRs accelerate carbon-cobalt bond homolysis by a factor of 1011. 2. Both class I and II RNRs are inactivated by the clinically active compounds gemcitabine and fluoromethylene cytidine di/triphosphates. Efforts will continue to elucidate the mechanism(s) of inactivation and the structures of the observed nucleotide radical intermediate(s). 3. Studies on the assembly mechanism of the class I RNR's diferric tyrosyl radical cofactor will continue using time resolved physical biochemical methods. Efforts to elucidate the structures of two new intermediates will be undertaken. In addition, further characterization of X, the direct precursor of the active cofactor will be undertaken. 4. Our long range goals are to understand the mechanism of cluster assembly of eucaryotic class I RNRs in vivo as well as in vitro and to understand the role of RNRs in both replication and repair. Toward these ends the yeast R1s (one of which is induced by DNA damage) and R2s will be engineered and expressed, the proteins purified, and antibodies generated. Efforts will be made to determine the location of these proteins within the yeast cell. Ultimately we would like to understand how the allosteric regulation of eucaryotic RNRs inside the cell control the fidelity of DNA replication, and the role of RNRs in check point control of the cell cycle.

描述：核糖核苷酸还原酶 (RNR) 催化速率决定 DNA生物合成中核苷酸向脱氧核苷酸转化的步骤。 它们在核酸代谢中的核心作用使它们成为最近 抗肿瘤和抗病毒药物设计的目标。 他们不寻常的金属 辅助因子及其在启动自由基依赖性核苷酸中的功能 通过形成硫基自由基的还原过程引起了人们的兴趣 无机化学家和有机化学家都一样。 1. 工作重点 了解长程耦合电子/质子转移的触发因素， I 类的两个亚基之间大约 35 埃 还原酶。 将继续努力调查 II 类 RNR 如何 将碳-钴键均裂加速 1011 倍。 2. 两类 I 和 II RNR 被临床活性化合物吉西他滨灭活 和氟亚甲基胞苷二磷酸/三磷酸。 将继续努力 阐明失活机制和结构 观察到的核苷酸自由基中间体。 3. 装配研究 I 类 RNR 的二铁酪氨酰自由基辅助因子的机制 继续使用时间分辨物理生化方法。 努力 将阐明两种新中间体的结构。 在 此外，进一步表征了活性物质的直接前体X 将进行辅助因子。 4. 我们的长期目标是了解 真核I类RNR在体内的簇组装机制以及 体外实验并了解 RNR 在复制和修复中的作用。 为了达到这些目的，酵母 R1（其中之一是由 DNA 损伤诱导的）和 R2 将被工程化和表达，蛋白质被纯化，抗体 生成的。 将努力确定这些蛋白质的位置 酵母细胞内。 最终我们想了解如何 细胞内真核 RNR 的变构调节控制着 DNA 复制的保真度，以及 RNR 在检查点控制中的作用 细胞周期。