IDENTIFICATION OF RADICAL INTERMEDIATES DURING INACTIVATION OF CLASS I RNRS

核糖核苷酸还原酶（RNR）在DNA中起着重要的作用 生物合成催化核苷酸转化为 脱氧核苷酸 I类和II类RNR是化学计量的 通过吉西他滨的二三磷酸盐（F2Cyt）灭活， 乙烯基氟胞苷（VFCyt）。 在I类酶中， 必需的酪氨酰基自由基伴随着新的 基于核苷酸的自由基。 新底物自由基的鉴定 通过CW-EPR光谱以前是复杂的光谱 稳定的酪氨酰基自由基与新的未鉴定的 激进，这就需要使用模棱两可的分析技术 比如光谱减法 最近，我们重新研究了EPR信号 在培养来自E.杆菌 2 '-叠氮基-2'-脱氧尿苷5 '-二磷酸（N3 UDP）和利用率高 灵敏度脉冲EPR谱在140 GHz。 电子自旋回波 在10 K下获得了具有良好信噪比的探测光谱 （> 100）。 脉冲EPR检测允许自由基物质的分离， 它们的特征在于， 放松时间 我们发现酪氨酰自由基的T， 在10 K时约为8 ms，比弛豫时间短得多 典型孤立有机自由基的速率（TI > 50 ms）。 我们的属性 短的弛豫时间与强交换相互作用 B2亚基中的必需二铁簇。 弛豫速率 在80 K左右，自旋回波随温度的升高而增强， 在短时间内，酪氨酰自由基的信号消失， 回波序列 我们证明，在80 K时， 酶抑制后仅由新的未知核苷酸组成 自由基和一个明确的模拟超精细和 g张量是可行的。 应用脉冲EPR技术研究 目前尚不清楚吉西他滨抑制RNR的机制 在研究中

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.