IDENTIFICATION OF RADICAL INTERMEDIATES DURING INACTIVATION OF CLASS I RNRS

核糖核苷酸还原酶(RNR)在DNA中起着至关重要的作用 生物合成催化核苷酸转化为 脱氧核苷酸。I类和II类RNR在化学计量上 用吉西他滨(F2Cyt)的二磷酸灭活和 乙烯基氟胞苷(VFCyt)。在I类酶中，部分损失 必需的酪氨酰自由基伴随着一种新的 以核苷酸为基础的自由基。新底物自由基的鉴定 通过CW-EPR谱以前是复杂的光谱 稳定的酪氨酸基与新的未知分子的重叠 激进，这就需要使用模棱两可的分析技术 就像谱减法一样。最近，我们重新研究了EPR信号 从大肠杆菌中培养出I类RNR后获得 2‘-叠氮-2’-脱氧尿苷5‘-二磷酸(N3UDP)，利用率高 140 GHz的灵敏度脉冲EPR谱。电子自旋回波 在10K下获得了良好的信噪比检测光谱 (&gt；100)。脉冲EPR检测允许在以下情况下分离自由基物种 它们的特征是本质上不同的T自旋晶格 放松时间。我们发现酪氨酸基的T，相当于 在10K时约为8ms，比弛豫时间短得多 典型孤立有机自由基的速率(TI&gt；50毫秒)。我们的属性 在较短的松弛时间内与强交流互动 必需的双铁素簇位于B2亚基。松弛速度 随着温度的升高而强烈增加，在约80K时，自旋回波 短刺激过程中酪氨酸基信号消失 回声序列。我们证明了在80K时所记录的EPR谱 酶抑制后仅由新的未知核苷酸组成 这是一个明确的模拟超精细和 G张量是可行的。脉冲电子顺磁共振在研究黄曲霉毒素中的应用 吉西他滨抑制RNR的机制目前尚不清楚 正在调查中。

Ribonucleotide Reductase (RNR) plays an essential role in DNA biosynthesis catalyzing the conversion of nucleotides to deoxynucleotides. Class I and II RNRs are stochiometrically inactivated by ditriphosphates of gemcitabine (F2Cyt) and vinylfluorocytidine (VFCyt). In the Class I enzymes, partial loss of the essential tyrosyl radical is accompanied by the formation of a new nucleotide based radical. Identification of the new substrate radical by CW-EPR spectroscopy was previously complicated by the spectral overlap of the stable tyrosyl radical with the new unidentified radical, which necessitated the use of ambiguous analysis techniques like spectral subtraction. Recently, we reinvestigated the EPR signal obtained after incubation of Class I RNR from E. coli with 2'-azido-2'-deoxyuridine 5'-diphosphate (N3UDP) and utilized high sensitivity pulsed EPR spectroscopy at 140 GHz. Electron-spin-echo detected spectra at 10 K were achieved with excellent signal-to-noise (> 100). Pulsed EPR detection allows separation of radical species if those are characterized by substantially different T, spin-lattice relaxation times. We found that T, of the tyrosyl radical amounts to about 8 ms at 10 K and is considerably shorter than the relaxation rate of typical isolated organic radicals (TI > 50 ms). We attribute the short relaxation time to a strong exchange interaction with the essential diiron cluster in the B2 subunit. The relaxation rate strongly increases with temperature and at about 80 K the spin-echo signal of the tyrosyl radical disappears during the short stimulated echo sequence. We demonstrated that at 80 K the EPR spectrum recorded after enzyme inhibition only consists of the new unknown nucleotide radical and that an unambiguous simulation of the hyperfine and g-tensors was feasible. Application of pulsed EPR to study the unknown mechanism of inhibition of RNR with gemcitabine is currently under investigation.