van der Donk W A, Yu G, Pérez L, Sanchez R J, Stubbe J, Samano V, Robins M J
Chemistry Department, Brigham Young University, 225 Eyring Science Center, P.O. Box 24672, Provo, Utah 84602-4672, USA.
Biochemistry. 1998 May 5;37(18):6419-26. doi: 10.1021/bi9729357.
Ribonucleotide reductases (RNRs) play a central role in replication and repair by catalyzing the conversion of nucleotides to deoxynucleotides. Gemcitabine 5'-diphosphate (F2CDP), the nucleoside of which was recently approved by the FDA for treatment of pancreatic cancer, is a potent mechanism-based inhibitor of class I and II RNRs. Inactivation of the Eschericia coli class I RNR is accompanied by loss of two fluorides and one cytosine. This RNR is composed of two homodimeric subunits: R1 and R2. R1 is the site of nucleotide reduction, and R2 contains the essential diferric-tyrosyl radical cofactor. The mechanism of inactivation depends on the availability of reductant. In the presence of reductant [thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH or dithiothreitol], inhibition results from R1 inactivation. In the absence of reductant with prereduced R1 and R2, inhibition results from loss of the essential tyrosyl radical in R2. The same result is obtained with C754S/C759S-R1 in the presence of TR/TRR/NADPH. In both cases, tyrosyl radical loss is accompanied by formation of a new stable radical (0.15-0.25 equiv/RNR). EPR studies in 2H2O, with [U-2H]R1, and examination of the microwave power saturation of the observed signal, indicate by process of elimination that this new radical is nucleotide-based. In contrast to all previously investigated 2'-substituted nucleotide inhibitors of RNR, inactivation is not accompanied by formation of a new protein-associated chromophore under any conditions. The requirement for reductant in the R1 inactivation pathway, the lack of chromophore on the protein, the loss of two fluoride ions, and the stoichiometry of the inactivation all suggest a unique mechanism of RNR inactivation not previously observed with other 2'-substituted nucleotide inhibitors of RNR. This unique mode of inactivation is proposed to be responsible for its observed clinical efficacy.
核糖核苷酸还原酶（RNRs）通过催化核苷酸转化为脱氧核苷酸，在复制和修复过程中发挥核心作用。吉西他滨5'-二磷酸（F2CDP），其核苷最近被美国食品药品监督管理局（FDA）批准用于治疗胰腺癌，是I类和II类RNRs的一种基于机制的强效抑制剂。大肠杆菌I类RNR的失活伴随着两个氟化物和一个胞嘧啶的丢失。这种RNR由两个同型二聚体亚基组成：R1和R2。R1是核苷酸还原的位点，R2含有必需的双铁 - 酪氨酸自由基辅因子。失活机制取决于还原剂的可用性。在有还原剂[硫氧还蛋白（TR）/硫氧还蛋白还原酶（TRR）/NADPH或二硫苏糖醇]存在的情况下，抑制作用源于R1失活。在没有还原剂且R1和R2预先还原的情况下，抑制作用源于R2中必需的酪氨酸自由基的丢失。在TR/TRR/NADPH存在的情况下，C754S/C759S - R1也会得到相同的结果。在这两种情况下，酪氨酸自由基的丢失都伴随着一种新的稳定自由基（0.15 - 0.25当量/RNR）的形成。在2H2O中使用[U - 2H]R1进行的电子顺磁共振（EPR）研究，以及对观察到的信号的微波功率饱和的检查，通过排除法表明这种新自由基是基于核苷酸的。与之前研究的所有RNR的2'-取代核苷酸抑制剂相比，在任何条件下失活都不会伴随着新的与蛋白质相关的发色团的形成。R1失活途径中对还原剂的需求、蛋白质上缺乏发色团、两个氟离子的丢失以及失活的化学计量比，都表明了一种RNR失活的独特机制，这是之前用其他RNR的2'-取代核苷酸抑制剂未观察到的。这种独特的失活模式被认为是其观察到的临床疗效的原因。
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