Molecular Mechanisms of Thioredoxin and Glutaredoxin as Hydrogen Donors for Mammalian S Phase Ribonucleotide Reductase
Molecular Mechanisms of Thioredoxin and Glutaredoxin as Hydrogen Donors for Mammalian S Phase Ribonucleotide Reductase
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DOI：
10.1074/jbc.m809338200
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发表时间：
2009-03-27
影响因子：
4.8
通讯作者：
Holmgren, Arne
中科院分区：
文献类型：
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作者：
Avval, Farnaz Zahedi;Holmgren, Arne
核糖核苷酸还原酶 (RNR) 催化脱氧核糖核苷酸合成中的限速步骤，这对 DNA 复制和修复至关重要。 S期哺乳动物细胞中的RNR包含含有氧化还原活性半胱氨酸残基的催化R1蛋白和含有酪氨酸自由基的R2蛋白的弱胞质复合物。每次酶转换都会在 R1 的活性位点产生一个二硫键，该二硫键被位于 C 端的穿梭二硫醇还原，留下二硫键被还原。用于还原的电子最终来自 NADPH，通过硫氧还蛋白还原酶和硫氧还蛋白 (Trx) 或谷胱甘肽还原酶、谷胱甘肽和谷氧还蛋白 (Grx)，但哺乳动物 RNR 的机制尚未阐明。使用重组小鼠 RNR，我们发现 Trx1 和 Grx1 具有相似的催化效率（kcat/Km）。在4mM GSH下，与Trx1相比，Grx1表现出更高的亲和力（表观Km值，0.18μM），Trx1表现出更高的表观k(cat)，表明其在S期DNA复制中的主要作用。令人惊讶的是，Grx 活性强烈依赖于 GSH 浓度（表观 Km 值，3 mM），并且尽管活性位点中只有一个半胱氨酸残基，Grx2 C40S 突变体仍具有活性。这证明了谷氧还蛋白催化的谷胱甘肽混合二硫键机制与硫氧还蛋白催化的二硫醇机制相反。这对于 DNA 修复的低 RNR 水平或具有高 RNR 且无或低 Trx 表达的肿瘤细胞来说可能是一个优势。我们的结果证明了哺乳动物和典型大肠杆菌 RNR 酶之间的机制差异，这可能为 R1 C 末端的非保守穿梭二硫醇序列提供解释。
Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in deoxyribonucleotide synthesis essential for DNA replication and repair. RNR in S phase mammalian cells comprises a weak cytosolic complex of the catalytic R1 protein containing redox active cysteine residues and the R2 protein harboring the tyrosine free radical. Each enzyme turnover generates a disulfide in the active site of R1, which is reduced by C-terminally located shuttle dithiols leaving a disulfide to be reduced. Electrons for reduction come ultimately from NADPH via thioredoxin reductase and thioredoxin (Trx) or glutathione reductase, glutathione, and glutaredoxin (Grx), but the mechanism has not been clarified for mammalian RNR. Using recombinant mouse RNR, we found that Trx1 and Grx1 had similar catalytic efficiency (kcat/Km). With 4mM GSH, Grx1 showed a higher affinity (apparent Km value, 0.18 mu M) compared with Trx1 which displayed a higher apparent k(cat), suggesting its major role in S phase DNA replication. Surprisingly, Grx activity was strongly dependent on GSH concentrations (apparent Km value, 3 mM) and a Grx2 C40S mutant was active despite only one cysteine residue in the active site. This demonstrates a GSH-mixed disulfide mechanism for glutaredoxin catalysis in contrast to the dithiol mechanism for thioredoxin. This may be an advantage with the low levels of RNR for DNA repair or in tumor cells with high RNR and no or low Trx expression. Our results demonstrate mechanistic differences between the mammalian and canonical Escherichia coli RNR enzymes, which may offer an explanation for the nonconserved shuttle dithiol sequences in the C terminus of the R1.