MECHANISM AND INHIBITION OF RIBONUCLEOTIDE REDUCTASES

核糖核苷酸还原酶的机制和抑制

• 批准号: 6385423
• 负责人: JOANNE STUBBE
• 金额: $ 36.79万
• 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/6385423
• 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）催化速率确定 核苷酸向脱氧核苷酸的DNA生物合成转化。 它们在核酸代谢中的核心作用使它们成为最近的 设计抗肿瘤和抗病毒药物的目标。 他们不寻常的金属 辅因子及其在启动自由基依赖性核苷酸方面的功能 通过形成硫基自由基的减少进程引起了人们的关注 无机和有机化学家。 1。努力将重点 了解远程耦合电子/质子传递的触发器， 在I类的两个亚基之间，大约35埃 还原酶。 努力将继续调查II级RNR如何 加速碳 - 铜键同型均匀分别为1011。2。 I和II RNR被临床活性化合物吉西他滨灭活 和荧光甲基胞苷二/三磷酸盐。 努力将继续 阐明失活的机制和结构 观察到的核苷酸自由基中间体。 3。关于组装的研究 I级RNR的差异酪酶自由基辅助因子的机制将 继续使用时间解决的物理生化方法。 努力 将阐明两个新中间体的结构。 在 另外，X的进一步表征，即活动的直接前体 将进行辅助因子。 4。我们的远距离目标是了解 Eucaryotic类I rnr的群集组装机理以及体内以及 体外，了解RNR在复制和修复中的作用。 朝向这些末端的酵母R1（其中之一是由DNA损伤诱导的），并且 R2将进行设计和表达，纯化蛋白质和抗体 生成。 将努力确定这些蛋白质的位置 在酵母细胞内。 最终，我们想了解 细胞控制中桉树RNR的变构调节 DNA复制的保真度，以及RNR在检查点控制中的作用 细胞周期。