Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit
Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit
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DOI：
10.1021/bi992206m
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发表时间：
2000-04-25
期刊：
影响因子：
2.9
通讯作者：
Lepoivre, M
中科院分区：
文献类型：
--
作者：
Guittet, O;Decottignies, P;Lepoivre, M
核糖核苷酸还原酶活性是 DNA 合成的速率限制，抑制该酶支持诱导型 NO 合酶的细胞抑制抗肿瘤作用。 I 类核糖核苷酸还原酶的小 R2 亚基含有活性所需的稳定自由基酪氨酸残基。该自由基被过氧亚硝酸盐破坏，过氧亚硝酸盐也会使蛋白质失活并诱导酪氨酸残基的硝化。在本报告中，通过紫外-可见光谱、质谱 (ESI-MS) 和胰蛋白酶肽测序鉴定了大肠杆菌 R2 蛋白中的硝化残基。质量分析允许检测蛋白质 R2 作为天然二聚体，每个亚基有两个铁簇。测量的质量为 87 032 Da，而计算值为 87 028 Da。过氧亚硝酸盐处理保留了非血红素铁中心和蛋白质的二聚体形式。每个大肠杆菌蛋白 R2 二聚体平均有两个硝基酪氨酸，在 400 μM 过氧亚硝酸盐下获得。 16 个酪氨酸中只有 3 个被硝化，包括自由基 Tyr122。尽管其处于激进状态，这应该有利于硝化，但埋藏的 Tyr122 并未以高产率被硝化，这可能是由于其可及性受到限制。 Tyr122 硝化和自由基损失的剂量反应曲线重叠。然而，蛋白质 R2 失活程度高于 ,, 的硝化，表明非保守 Tyr62 和 Tyr289 的硝化可能对于 Tyr122 过氧亚硝酸盐介导的大肠杆菌蛋白质 R2 抑制也很重要。
Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 mu M peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of,,, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for Tyr122 peroxynitrite-mediated inhibition of E. coli protein R2.