Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307, USA.
Biochemistry. 2011 Mar 1;50(8):1403-11. doi: 10.1021/bi101319v. Epub 2011 Feb 8.
Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y(122)(•)) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C(439)), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y(122) → W(48) → Y(356) in β2 to Y(731) → Y(730) → C(439) in α2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F(3)Y (F(3)Y) was site-specifically incorporated in place of Y(356) in β2 and 3-NH(2)Y (NH(2)Y) in place of Y(731) and Y(730) in α2. A pH-rate profile with F(3)Y(356)-β2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F(3)Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH(2)Y(731(730))-α2, β2, CDP, and ATP resulted in detection of NH(2)Y radical (NH(2)Y(•)) intermediates capable of dNDP formation. In this study, the reaction of F(3)Y(356)-β2, α2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F(3)Y(356)-β2, CDP, and ATP has also been examined with NH(2)Y(731)-α2 (or NH(2)Y(730)-α2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH(2)Y(•) formation that support a change in the rate-limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway.
大肠杆菌核糖核苷酸还原酶是一种α2β2 复合物，可催化核苷 5'-二磷酸 (NDP) 转化为脱氧核苷酸 (dNDP)。NDP 还原的活性部位位于α2 中，起始将自由基转移到α2 中活性部位半胱氨酸 (C(439)) 的必需的双核铁 - 酪氨酸自由基 (Y(122)(•)) 辅助因子，距离 35 Å，位于β2 中。氧化被认为涉及通过芳香族氨基酸 (Y(122)→W(48)→β2 中的 Y(356)→Y(731)→Y(730)→α2 中的 C(439)) 和可逆质子偶联电子转移 (PCET) 的跳跃机制。最近，2,3,5-F(3)Y (F(3)Y) 被特异性地取代β2 中的 Y(356)和α2 中的 3-NH(2)Y (NH(2)Y) 取代 Y(731)和 Y(730)。带有 F(3)Y(356)-β2 的 pH 速率曲线表明，随着 pH 值升高，RNR 的速率决定步骤可以从构象变化改变为 PCET，并且通过途径中紧邻它的残基改变 F(3)Y 氧化的驱动力负责这种变化。使用 NH(2)Y(731(730))-α2、β2、CDP 和 ATP 的研究导致检测到能够形成 dNDP 的 NH(2)Y 自由基 (NH(2)Y(•)) 中间体。在这项研究中，通过停流 (SF) 吸收和快速冷冻淬火电子顺磁共振波谱研究了 F(3)Y(356)-β2、α2、CDP 和 ATP 的反应，但未能揭示任何自由基中间体。F(3)Y(356)-β2、CDP 和 ATP 的反应也已在 pH 6.5 至 9.2 下通过 SF 动力学用 NH(2)Y(731)-α2（或 NH(2)Y(730)-α2）进行了检查，并表现出 NH(2)Y(•)形成的速率常数，支持在升高的 pH 值下限速步骤的变化。这些结果连同动力学模拟为未来检测途径中的自由基中间体提供了指导。
