Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases.
Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases.
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DOI：
10.1021/ja201640n
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发表时间：
2011-06-22
影响因子：
15
通讯作者：
Stubbe, JoAnne
中科院分区：
文献类型：
--
作者：
Minnihan, Ellen C.;Seyedsayamdost, Mohammad R.;Uhlin, Ulla;Stubbe, JoAnne
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2′-脱氧核苷酸（dNDPs）。该反应是由α2中的活性半胱氨酸（C439）被β2中稳定的异酪氨酸自由基（Y122•）辅因子瞬间氧化而引发的。该氧化过程是通过质子耦合电子转移（PCET）机制在35 Å上通过特定的残基途径发生的：β中的Y122•→W48→Y356到α中的Y731→Y730→C439。为了研究这一过程的细节，3-氨基酪氨酸（NH2Y）被特异性地掺入代替β的Y356。结果表明，得到的蛋白Y356NH2Y-β2和先前生成的蛋白Y731NH2Y-α2和Y730NH2Y-α2 （NH2Y-RNRs）在第二亚基、底物(S)和变构效应物(E)存在的情况下催化dNDP的产生，周转数为0.2-0.7 S−1。通过三种不同的方法获得的证据表明，催化活性是NH2Y-RNRs固有的，而不是wt酶共纯化的结果。3-氨基酪氨酸自由基（NH2Y•s）在356、731和730位点的形成动力学用所有s /E对进行了测量。在所有情况下，NH2Y•的形成都是双相的（kfast为9-46 s−1，kslow为1.5-5.0 s−1），并且在动力学上能够成为核苷酸还原的中间体。慢相被认为报告了NH2Y•形成的构象门控，而~0.5 s−1的kcat被认为与正向PCET过程中NH2Y•对途径上后续氨基酸的限速氧化有关。对Y730NH2Y-α2和Y731NH2Y-α2的x射线晶体结构进行了求解，结果表明，与wt-α2相比，Y730NH2Y-α2的结构变化很小。根据这些数据，提出了PCET沿自由基传播路径的动力学模型。
Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5´-diphosphates (NDPs) to 2´-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C439) in α2 by a stable diferric tyrosyl radical (Y122•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y122•→ W48→ Y356 in β to Y731→ Y730→ C439 in α. To study the details of this process, 3-aminotyrosine (NH2Y) has been site-specifically incorporated in place of Y356 of β. The resulting protein, Y356NH2Y-β2, and the previously-generated proteins Y731NH2Y-α2 and Y730NH2Y-α2 (NH2Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2–0.7 s−1. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH2Y-RNRs and not the result of co-purifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH2Y•s) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH2Y• formation is biphasic (kfast of 9–46 s−1 and kslow of 1.5–5.0 s−1) and kinetically-competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational-gating of NH2Y• formation, while the kcat of ~0.5 s−1 is proposed to be associated with rate-limiting oxidation by NH2Y• of the subsequent amino acid on the pathway during forward PCET. The Xray crystal structures of Y730NH2Y-α2 and Y731NH2Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.
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影响因子：
2.9
作者：
Artin, Erin;Wang, Jun;Lohman, Gregory J. S.;Yokoyama, Kenichi;Yu, Guixue;Griffin, Robert G.;Bar, Galit;Stubbe, JoAnne
通讯作者：
Stubbe, JoAnne
肽和氨基酸类似物中酪氨酸苯氧基和色氨酸吲哚基的电化学性质
影响因子：
--
作者：
DEFELIPPIS, MR;MURTHY, CP;KLAPPER, MH
通讯作者：
KLAPPER, MH
影响因子：
2.9
作者：
Seyedsayamdost, Mohammad R.;Yee, Cyril S.;Stubbe, JoAnne
通讯作者：
Stubbe, JoAnne
影响因子：
15
作者：
JOVANOVIC, SV;STEENKEN, S;SIMIC, MG
通讯作者：
SIMIC, MG
影响因子：
15
作者：
Seyedsayamdost, Mohammad R.;Stubbe, JoAnne
通讯作者：
Stubbe, JoAnne