Ribonucleotide Reductases: Structure and Function

描述（由申请人提供）：核糖核苷酸还原酶（RNR）在所有生物体中催化核苷酸转化为脱氧核苷酸，提供DNA复制和修复所需的单体前体。RNR作为一个范例，用于理解自然界如何利用蛋白质和核苷酸自由基的增强反应性，并进行精确控制，以产生脱氧核苷酸。RNR在核酸代谢中的核心作用使其成为临床上使用的两种药物的成功靶点：2 '，2”-二氟-2'-脱氧胞苷（吉西他滨）和羟基脲。这两种化合物干扰RNR的自由基化学，其细节正在本提案中进行研究。I类RNR需要二铁-酪氨酰基自由基（Y（））辅因子用于催化。它的功能是启动前所未有的长距离质子耦合电子转移（PCET），自由基传播步骤，超过35 A的距离。这个过程发生在RNR的亚基：R1和R2之间。方法来测量这个距离与位点特异性连接的探针和PELDOR和DQC方法。描述了使用非天然氨基酸（FnY，x = 1-4）通过内含肽介导的蛋白质连接或邻位tRNA/tRNA合成酶对结合体内翻译机器置于每个亚基中来研究PCET的方法。本文介绍了用光触发PCET的方法，以检测瞬时氨基酸自由基中间体。正常还原过程的研究，基于抑制剂的机制，自由基传播和调节的特异性和核苷酸还原速率，都需要了解R1的四级结构及其与R2的相互作用。生物物理方法（超离心，动态光散射，停止流动荧光）使用特定位置放置的荧光和交联剂在R1和R2的，以解决这个问题。这些研究对于实现我们的长期目标至关重要：了解细胞如何生物合成和维持RNR活性所必需的diferric-Y（辅因子），并定量了解体内控制dNTP池的复杂调控层。急诊coli RNR和S.酿酒酵母和人类RNR是我们努力的重点。

DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms providing the monomeric precursors required for DNA replication and repair. The RNRs serve as a paradigm for understanding how Nature has harnessed the enhanced reactivity of protein and nucleotide free radicals with exquisite control to produce deoxynucleotides. The central role of RNRs in nucleic acid metabolism has made them the successful target of two drugs used clinically: 2', 2"-difluoro-2'-deoxycytidine (gemzar) and hydroxyurea. Both compounds interfere with the radical chemistry of the RNRs, the details of which are being investigated in the present proposal. The class I RNRs, require a diferric-tyrosyl radical (Y() cofactor for catalysis. Its function is to initiate the unprecedented long range proton coupled electron transfer (PCET), the radical propagation step, over a 35 A distance. This process occurs between the subunits of the RNR: R1 and R2. Methods to measure this distance with site-specifically attached probes and PELDOR and DQC methods are presented. Methods to study PCET using unnatural amino acids (FnYs with x = 1-4) placed into each subunit by intein mediated protein ligations or orthologous tRNA/tRNA synthetase pairs in conjunction with the translational machinery in vivo are described. Methods to trigger the PCET with light are presented in an effort to detect transient amino acid radical intermediates. Studies of the normal reduction process, mechanism based inhibitors, radical propagation and regulation of the specificity and rate of nucleotide reduction, all require an understanding of the quaternary structure of R1 and its interaction with R2. Biophysical methods (ultracentrifugation, dynamic light scattering, stopped flow fluorescence) using site- specifically placed fluorescent and cross-linking agents within R1 and R2 are presented to address this issue. These studies are essential for achieving our long range goals: to understand how cells biosynthesize and maintain the diferric-Y( cofactor essential for RNR activity and to understand quantitatively the complex layers of regulation that control dNTP pools in vivo. The E. coli RNR and the S. cerevisiae and human RNRs are the focus of our efforts.