Insight into the mechanism of inactivation of ribonucleotide reductase by gemcitabine 5'-diphosphate in the presence or absence of reductant.
Insight into the mechanism of inactivation of ribonucleotide reductase by gemcitabine 5'-diphosphate in the presence or absence of reductant.
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DOI：
10.1021/bi901590q
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发表时间：
2009-12-15
期刊：
影响因子：
2.9
通讯作者：
Stubbe, JoAnne
中科院分区：
文献类型：
--
作者：
Artin, Erin;Wang, Jun;Lohman, Gregory J. S.;Yokoyama, Kenichi;Yu, Guixue;Griffin, Robert G.;Bar, Galit;Stubbe, JoAnne
吉西他滨5′-二磷酸（F2CDP）是核糖核苷酸还原酶（RNR）的强效抑制剂，RNR是将核苷酸（NDP）转化为脱氧核苷酸的酶，对DNA复制和修复至关重要。急诊大肠杆菌RNR是一种α2β2复合物，当与一当量的F2CDP孵育时，催化两种氟化物和胞嘧啶的释放，同时使酶失活。在存在还原剂（硫氧还蛋白/硫氧还蛋白还原酶/NADPH或DTT）的情况下，酶失活是由于其用F2CDP的糖共价标记α（单标记/α2β2）。未煮沸样品的灭活RNR的SDS PAGE分析显示，α以87 kDa和110 kDa蛋白的比例迁移，比例为0.6：0.4。当省略还原剂时，RNR由于基本酪氨酰基自由基的损失和新自由基的形成而失活。在存在或不存在还原剂的情况下对C225 S-α的失活研究表明，在不存在还原剂的情况下，它的行为类似于wt-RNR。失活的C225 S-α作为87 kDa蛋白迁移，未共价修饰。C225是RNR活性位点中的半胱氨酸之一，其提供还原当量以产生dNDP。为了确定新形成的自由基，[1′-2H] F2CDP与wt-和C225 S-RNR通过9和140 GHz EPR光谱进行了研究。这些研究表明，新的自由基是核苷酸衍生的，g值为gx 2.00738，戈伊2.00592，gz 2.00230，相对于[1′-1 H] F2CDP具有改变的超精细相互作用（表观三重态塌陷为双重态）。EPR特征与我们最近报道的CDP和E441 Q-RNR产生的核苷酸自由基非常相似。
Gemcitabine 5′-diphosphate (F2CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The E. coli RNR, an α2β2 complex, when incubated with one equivalent of F2CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of α with the sugar of F2CDP (one-label/α2β2). SDS PAGE analysis of the inactivated RNR without boiling of the sample reveals that α migrates as an 87 kDa and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-α in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-α migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR’s active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1′-2H] F2CDP was studied with wt- and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is nucleotide derived with g values of gx 2.00738, gy 2.00592, gz 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1′-1H] F2CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR.
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影响因子：
2.9
作者：
CLIMENT, I;SJOBERG, BM;HUANG, CY
通讯作者：
HUANG, CY
DOI：
10.1080/07328319708006168
发表时间：
1997-01-01
期刊：
NUCLEOSIDES & NUCLEOTIDES
影响因子：
--
作者：
Plunkett, W;Huang, P;Gandhi, V
通讯作者：
Gandhi, V
影响因子：
2.2
作者：
Bennati, M;Farrar, CT;Griffin, RG
通讯作者：
Griffin, RG
影响因子：
3.6
作者：
Hsu, CH;Liou, JY;Cheng, YC
通讯作者：
Cheng, YC
影响因子：
2.9
作者：
Silva, DJ;Stubbe, J;Robins, MJ
通讯作者：
Robins, MJ