Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example.
Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example.
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DOI：
10.1021/jacs.6b03605
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发表时间：
2016-06-29
影响因子：
15
通讯作者：
Stubbe J
中科院分区：
文献类型：
--
作者：
Oyala PH;Ravichandran KR;Funk MA;Stucky PA;Stich TA;Drennan CL;Britt RD;Stubbe J
氟化酪氨酸（FnY’s，n = 2和3）已被位点特异性地掺入E. coliIa类核糖核苷酸还原酶（RNR）。詹氏Y-tRNA合成酶/tRNA对。Ia类RNR需要四个氧化还原活性Y，β亚基中的稳定Y自由基（Y·）（E.大肠杆菌），和三个瞬时氧化的Y（β中的356和α中的731和730），以启动自由基依赖的核苷酸还原过程。FnY（3，5; 2、3;二、三、五;和2，3，6）取代Y_（122-β）的掺入，并报道了每种含二铁团簇的β的X射线结构，并与相同条件下结晶的wt-β_2进行了比较。必需的差铁-FnY·辅因子由apo FnY-β2、Fe 2+和O2自组装产生101 Y·/β2和103 Fe 3 +/β2。FnY·在核苷酸还原中是稳定和有活性的，其活性为wt-β2的5%至85%。每个FnY·-β2都用9和130 GHz电子顺磁共振和高场电子核双共振谱表征。与19 F核相关的超精细相互作用提供了每个FnY·的独特特征，这些特征很容易与未标记的Y·区分开来。非生物FnY的pKa（6.4至7.8）和还原电位（相对于Y在pH 7.5时为−30至+130 mV）的可变性提供了酶促反应的探针，该探针被提议涉及Y·的催化作用，并研究了氧化还原活性蛋白中跳跃Y·的重要性和身份，该蛋白被提议保护它们免受非偶联自由基化学的影响。
Fluorinated tyrosines (FnY’s, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y’s, a stable Y radical (Y·) in the β subunit (position 122 in E. coli), and three transiently oxidized Y’s (356 in β and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-β and the X-ray structures of each resulting β with a diferric cluster are reported and compared with wt-β2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-β2, Fe2+, and O2 to produce ∼1 Y·/β2 and ∼3 Fe3+/β2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each FnY·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the 19F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·’s. The variability of the abiotic FnY pKa’s (6.4 to 7.8) and reduction potentials (−30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·’s in catalysis and to investigate the importance and identity of hopping Y·’s within redox active proteins proposed to protect them from uncoupled radical chemistry.