Eriksson S, Caras I W, Martin D W
Proc Natl Acad Sci U S A. 1982 Jan;79(1):81-5. doi: 10.1073/pnas.79.1.81.
The protein M1 subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity of the enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein M1 from mouse T-lymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0 degrees C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein M1 isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism.
核糖核苷酸还原酶的蛋白质M1亚基至少包含两个变构核苷酸结合位点，这些位点控制着该酶将核糖核苷酸还原为DNA合成所需的脱氧核糖核苷酸的能力。在0℃下，于dTTP存在的情况下进行紫外线照射后，观察到从小鼠T淋巴瘤（S49）细胞中部分纯化的蛋白质M1的直接光亲和标记。每个亚基核苷酸的相对摩尔掺入率为4 - 8%。竞争实验表明，dTTP与一个变构结构域结合，该结构域在遗传和动力学上被定义为该酶的底物特异性位点。从抗胸苷突变细胞系中分离出的一种改变的蛋白质M1显示dTTP的光掺入显著减少，这与它的CDP还原酶活性对dTTP的反馈抑制具有抗性这一事实相一致。还发现了用嘧啶和嘌呤核苷酸对其他几种蛋白质进行特异性光标记，表明直接光亲和标记在研究参与核苷酸和核酸代谢的酶方面具有普遍用途。
