Gemcitabine 5′-triphosphate is a stoichiometric mechanism-based inhibitor of Lactobacillus leichmannii ribonucleoside triphosphate reductase:: Evidence for thiyl radical-mediated nucleotide radical formation
Gemcitabine 5′-triphosphate is a stoichiometric mechanism-based inhibitor of Lactobacillus leichmannii ribonucleoside triphosphate reductase:: Evidence for thiyl radical-mediated nucleotide radical formation
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DOI：
10.1021/bi972934e
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发表时间：
1998-04-21
期刊：
影响因子：
2.9
通讯作者：
Robins, MJ
中科院分区：
文献类型：
--
作者：
Silva, DJ;Stubbe, J;Robins, MJ
来自莱希曼乳杆菌的核糖核苷三磷酸还原酶 (RTPR) 利用腺苷钴胺素并催化核苷三磷酸转化为脱氧核苷三磷酸。一当量的 2',2'-二氟-2'-脱氧胞苷 5'-三磷酸 (F(2)dCTP) 可快速灭活 RTPR。对反应产物的分析表明，失活伴随着两个氟离子和 0.84 当量 5'-脱氧腺苷的释放，以及 1 当量的 Corrin 与 RTPR 活性位点半胱氨酸残基的共价连接。没有检测到胞嘧啶释放。使用内切蛋白酶 Glu-C 对 Corrin 标记的 RTPR 进行蛋白水解，并在 pH 5.8 下进行肽图分析，结果表明 C419 主要被修饰。通过停流（SF）紫外可见光谱和快速冷冻猝灭（RFQ）电子顺磁共振（EPR）光谱检查了失活动力学。监测 Delta A(525) nm 表明 cob(II)alamin 形成的表观 k(obs) 为 50 s(-1)，仅比用胞苷 5'-三磷酸 (CTP) 进行的类似实验慢 2.5 倍。因此，相同的反应混合物在 22 毫秒至 30 秒的时间内被淬灭，并通过 EPR 光谱进行检查。在早期时间点，EPR 谱类似于与钴 (II) 胺胺偶联的硫基自由基交换。从 22 到 255 ms，总自旋浓度保持不变，为 1.4 自旋/RTPR，是 SF 测定的 cob(II)alamin 量预测值的两倍。然而，随着时间的推移，硫基自由基-cob(rr)alamin 的信号消失，并出现了在 g = 2.33 处具有广泛特征的新信号和在 g = 2.00 处具有尖锐特征的新信号，表明形成了 cob(II)alamin 和仅具有偶极相互作用的基于核苷酸的自由基。这些研究被解释为支持基于 RTPR 的硫基自由基可以产生基于核苷酸的自由基的提议。
Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii utilizes adenosylcobalamin and catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates, One equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate, F(2)dCTP, rapidly inactivates RTPR. Analysis of the reaction products reveals that inactivation is accompanied by release of two fluoride ions and 0.84 equiv of 5'-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detected. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modified. The kinetics of the inactivation have been examined by stopped-flow (SF) UV-vis spectroscopy and rapid freeze quench (RFQ) electron paramagnetic resonance (EPR) spectroscopy. Monitoring Delta A(525) nm shows that cob(II)alamin is formed with an apparent k(obs) of 50 s(-1), only 2.5-fold slower than a similar experiment carried out with cytidine 5'-triphosphate (CTP). The same reaction mixture was thus quenched at times from 22 ms to 30 s and examined by EPR spectroscopy. At early time points the EPR spectrum resembled a thiyl radical exchange coupled to cob(II)alamin. From 22 to 255 ms the total spin concentration remained unchanged at 1.4 spins/RTPR, twice that predicted by the amount of cob(II)alamin determined by SF. However, with time the signal attributed to the thiyl radical-cob(rr)alamin disappears and new signal(s) with broad feature(s) at g = 2.33 and a sharp feature at g = 2.00 appeared, suggesting formation of cob(II)alamin and a nucleotide-based radical with only dipolar interactions. These studies have been interpreted to support the proposal that an RTPR-based thiyl radical can give rise to a nucleotide-based radical.