AZIDO INHIBITOR OF RIBONUCLEOTIDE REDUCTASE

2 '-叠氮基-2'-脱氧尿苷-5 '-二磷酸（N3 UDP）先前已 已被证明是基于化学计量机制的RNR抑制剂。 RNR的失活伴随着酪氨酰自由基的损失， R2亚基伴随着新氮中心的形成 激进 这种稳定自由基的X波段EPR谱 显示出-25 G的三重态超精细相互作用，其产生于以下之一： N3 UDP的叠氮部分的三个氮和一个双态 6.3高斯的超精细相互作用， 从一个质子。 高频（139.5 GHz）EPR波谱研究 这个氮中心的自由基已经解决了三个主要的 g值：g 11 = 2.01557、922 = 2.00625和933 = 2.00209。 在 此外，氮超精细分裂沿着933被解析（A33= 3 1.0 G）和上限（- 5 G），可设置在A I和A 22上 . 这些g和A的比较 模型系统的值， 文献提出了基团X-N-S-CH 2-的结构， 其中SCH 2是RI的半胱氨酸残基的一部分，并且X是 非质子化的硫、氧或碳部分。 使用E。杆菌 半胱氨酸营养缺陷型菌株，含有核苷酸 还原酶基因允许制备[p-2 H]-半胱氨酸标记的RNR。 将该同位素标记的蛋白质与产生的N3 UDP孵育 没有6.3G超精细分裂的自由基信号， 这表明这种相互作用与来自 - 所提出的结构的S-CH 2-组分。 这些结果证实 以氮为中心的基团共价连接到 RNR RI亚基的半胱氨酸，可能是C225。 定点 用各种RI突变体进行诱变研究，其中每种 半胱氨酸（439，462，754和759）转化为丝氨酸，表明X 不能是取代的硫。 一种以氮为中心的结构 提出了一个自由基，其中X衍生自3 ′-酮基-2 ′-脱氧尿苷 5 '-二磷酸，N3 UDP使RNR失活的中间体。 具体地说，X被认为是 脱氧核糖部分。

2'-Azido-2'-deoxyuridine- 5'-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism based inhibitor of RNR. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen centered radical. The Xband EPR spectrum of this stable radical species exhibits a triplet hyperfine interaction of -25 G arising from one of the three nitrogens of the azide moiety of N3UDP and a doublet hyperfine interaction of 6.3 Gauss which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen centered radical have resolved the three principal g-values: g I I = 2.01557, 922 2.00625, and 933 = 2.00209. In addition, the nitrogen hyperfine splitting along 933 is resolved (A33= 3 1.0 G) and upper limits (- 5 G) can be placed on both A I , and A 22 . Comparison of these g and A values with those of model systems in the literature suggests a structure for the radical, X-N--S-CH2-, in which SCH2 is part of a cysteine residue of RI and X is either a non-protonated sulfur, oxygen or carbon moiety. Use of an E. coli strain that is auxotrophic for cysteine and contains the nucleotide reductase gene allowed [p-2H]-cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the -S-CH2-component of the proposed structure. These results establish that the nitrogen centered radical is covalently attached to a cysteine, probably C225, of the RI subunit of RNR. Site-directed mutagenesis studies with a variety of RI mutants in which each cysteine (439, 462, 754 & 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen centered radical is proposed in which X is derived from 3'-keto-2'-deoxyuridine 5'-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the Y-hydroxyl oxygen of the deoxyribose moiety.