Mono-, di-, tri-, and tetra-substituted fluorotyrosines: New probes for enzymes that use tyrosyl radicals in catalysis
Mono-, di-, tri-, and tetra-substituted fluorotyrosines: New probes for enzymes that use tyrosyl radicals in catalysis
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DOI：
10.1021/ja055926r
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发表时间：
2006-02-08
影响因子：
15
通讯作者：
Stubbe, J
中科院分区：
文献类型：
--
作者：
Seyedsayamdost, MR;Reece, SY;Stubbe, J
合成了一系列N-酰化羧胺类氟酪氨酸类似物[Ac-3-FY-NH2，Ac-3，5-F2Y-NH2，Ac-2，3-F2Y-NH2，Ac-2，3，5-F3Y-NH2，Ac-2，3，6-F3Y-NH2，Ac-2，3，6-F3Y-NH2和Ac-2，3，5，6-F4Y-NH2]，以探讨FNY中心点S在小分子和蛋白质中可能存在的详细反应机理(S)。这些Ac-FNY-NH2衍生物的pK(A)范围从5.6到8.4，在大多数蛋白质(6-9)可及的pH范围内的还原电位范围为320 mV。用密度泛函电子结构方法计算了4-苯甲酰基-L-苯丙氨基-F(N)YS的甲酯在pH为4时的电子结构，得到了FNY中心点S的吸收光谱，并通过溶液在pH 11和77K下的紫外光分解测定了每个FNY中心点的EPR谱。系统地改变pKA和自由基还原势的能力，以及具有明显吸收和EPR特征的监测自由基形成的装置，确定F(N)Ys在研究涉及酪氨酸的生物电荷传输机制方面将是有用的。为了证明氟酪氨酸方法在揭示复杂生物体系中电荷传输方面的有效性，我们报道了核糖核苷酸还原酶(RNR)R2亚基中3-氟酪氨酸(3-FY)对酪氨酸的整体取代，并给出了EPR谱及其对3-FY122中心点的模拟。在配对的论文中，我们展示了FnYS的效用，通过将它们整合到大肠杆菌RNR的R2亚单位的356位位置上，从而深入了解生物系统中酪氨酸氧化的机制。
A set of N-acylated, carboxyamide fluorotyrosine (FnY) analogues [Ac-3-FY-NH2, Ac-3,5-F2Y-NH2, Ac-2,3-F2Y-NH2, Ac-2,3,5-F3Y-NH2, Ac-2,3,6-F3Y-NH2 and Ac-2,3,5,6-F4Y-NH2] have been synthesized from their corresponding amino acids to interrogate the detailed reaction mechanism(s) accessible to FnY center dot S in small molecules and in proteins. These Ac-FnY-NH2 derivatives span a pK(a) range from 5.6 to 8.4 and a reduction potential range of 320 mV in the pH region accessible to most proteins (6-9). DFT electronic-structure calculations capture the observed trends for both the reduction potentials and pK(a)s. Dipeptides of the methyl ester of 4-benzoyl-L-phenylalanyl-F(n)Ys at pH 4 were examined with a nanosecond laser pulse and transient absorption spectroscopy to provide absorption spectra of FnY center dot S. The EPR spectrum of each FnY center dot has also been determined by UV photolysis of solutions at pH 11 and 77 K. The ability to vary systematically both pKa and radical reduction potential, together with the facility to monitor radical formation with distinct absorption and EPR features, establishes that F(n)Ys will be useful in the study of biological charge-transport mechanisms involving tyrosine. To demonstrate the efficacy of the fluorotyrosine method in unraveling charge transport in complex biological systems, we report the global substitution of tyrosine by 3-fluorotyrosine (3-FY) in the R2 subunit of ribonucleotide reductase (RNR) and present the EPR spectrum along with its simulation of 3-FY122 center dot. In the companion paper, we demonstrate the utility of FnYS in providing insight into the mechanism of tyrosine oxidation in biological systems by incorporating them site-specifically at position 356 in the R2 subunit of Escherichia coli RNR.