AZIDO INHIBITOR OF RIBONUCLEOTIDE REDUCTASE

2‘-叠氮-2’-脱氧尿苷-5‘-二磷酸(N3UDP) 已被证明是一种基于化学计量机理的RNR抑制剂。 RNR的失活伴随着酪氨酸自由基的丢失 R2亚基伴随着新的氮中心的形成 激进的。该稳定自由基物种的X波段电子顺磁共振波谱 表现出-25G的三重态超精细相互作用，产生于 N3UDP叠氮部分的三个氮基和一个二重态 已提出的6.3高斯超精细相互作用 来自一个质子。高频(139.5 GHz)电子顺磁共振波谱研究 这个以氮为中心的自由基已经解决了三个主要问题 G值：Gi=2.01557、922 2.00625和933=2.00209。在……里面 此外，解决了沿933的氮超精细分裂(A33= 3 1.0G)和上限(-5G)可以设置在A I和A 22上 。这些g值和A值与模型系统的g值和A值在 文献中提出了一种结构，X-N-S-CH2-，in 其中SCH2是RI的半胱氨酸残基的一部分，而X是 非质子化的硫、氧或碳部分。大肠杆菌的使用 一种对半胱氨酸有营养缺乏症且含有核苷酸的菌株 还原酶基因可以制备[p-2H]-半胱氨酸标记的RNR。 该同位素标记蛋白与生产的N3UDP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 -S-CH2-拟议结构的组成部分。这些结果证实了 以氮为中心的基团共价连接到一个 RNR的RI亚基的半胱氨酸，可能是C225。站点定向 不同RI突变体的诱变研究 半胱氨酸(439,462,754和759)被转化为丝氨酸，揭示了X 不能被取代的硫磺。一种以氮为中心的结构 提出了X由3‘-酮-2’-脱氧尿苷衍生的基团 5‘-二磷酸，N3UDP使RNR失活的中间产物。 具体地说，X被建议为Y-羟基氧 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.