ASSEMBLY OF THE R2 COFACTOR OF RIBONUCLEOTIDE REDUCTASE

核糖核苷酸还原酶是一种负责将 从核糖核苷酸到脱氧核糖核苷酸。因为这是一个至关重要的过程 DNA合成，该酶是抗癌和 抗病毒药物疗法。这项研究的长期目标是 了解该酶的催化机理，以便 开发有效的临床治疗方法。这项工作的重点是 确定辅因子在大肠杆菌中的组装机制和 哺乳动物的酶。酶R2亚基的催化辅因子 是一个离酪氨酸基很近的二铁簇合物。这 自由基是启动长程电子转移到 底物还原的第二亚单位(RI)。到目前为止，有几个 在R2辅因子的组装中已经确定了中间产物 来源于脱脂蛋白、Fe2+和02。这项工作将继续进行结构 中间体X的表征可以被困在 快速冷冻冷冻法在大肠杆菌酶中的组装工艺 光谱(EPR、穆斯堡尔、拉曼和EXAFS)。通过更改 组装过程中的条件(pH、温度、突变体或反应物)，即 希望能够积累一种可能的Fe3+-Peroxo物种。最后， 这些对特性良好的大肠杆菌酶的研究将会扩大。 对研究较少的哺乳动物的酶，因为很可能有物种 临床上可能存在的组装过程中的依赖差异 切合实际。

Ribonucleotide reductase is the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides. As this is a crucial process in DNA synthesis, the enzyme is a favored target for anticancer and antiviral drug therapies. The long term goal of this research is to understand the catalytic mechanism of the enzyme for purposes of developing effective clinical treatments. This work focuses on determining the mechanism for cofactor assembly in the E. coli and mammalian enzymes. The catalytic cofactor of the R2 subunit of the enzyme is a diferric cluster in close proximity of a tyrosine radical. This radical is essential for initiating long range electron transfer to the second subunit (RI) where the substrate is reduced. Thus far, several intermediates have been identified in the assembly of the R2 cofactor from apoprotein, Fe2+ and 02. This work will continue the structural characterization of the intermediate X that can be trapped in the assembly process in E. coli enzyme using rapid freeze quench spectroscopies (EPR, Mossbauer, Raman and EXAFS). By altering the conditions during assembly (pH, temperature, mutants, or reactants), I hope to be able to accumulate a putative Fe3+-peroxo species. Finally, these studies on the well characterized E. coli enzyme will be extended to the lesser studied mammalian enzyme, as there are likely to be species dependent differences in the assembly process that may be clinically relevant.