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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Structure/function correlations among coupled binuclear copper proteins through spectroscopic and reactivity studies of NspF
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Structure/function correlations among coupled binuclear copper proteins through spectroscopic and reactivity studies of NspF

机译:通过NspF的光谱和反应性研究,偶联的双核铜蛋白之间的结构/功能相关性

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摘要

The terminal step of 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis is performed by NspF, a mono-oxy-genase that converts o-aminophenols to the corresponding nitroso product (hydroxyanilinase activity). Previous biochemical characterization of the resting form of NspF suggested that this enzyme belonged to the coupled binuclear copper enzyme (CBC) family. Another member of this enzyme family, tyrosmase, is able to mono-oxygenate monophenols (monophenolase activity) but not o-aminophenols. To gain insight into the unique reactivity of NspF, we have generated and characterized the oxy form of its active site. The observation of spectral features identical to those of oxy-tyrosinase indicates that oxy-NspF contains a Cu_2O_2 core where peroxide is coordinated in a μ-η~2: η~2 mode, confirming that NspF is a CBC enzyme. This oxy form is found to react with monophenols, indicating that, like tyrosinase, NspF also possesses monophenolase activity. A comparison of the two electrophilic mechanisms for the monophenolase and hydroxyanilinase activity indicates a large geometric change between their respective transition states. The potential for specific interactions between the protein pocket and the substrate in each transition state is discussed within the context of the differential reactivity of this family of enzymes with equivalent μ-η~2:η~2 peroxy bridged coupled binuclear copper active sites.
机译:村山链霉菌中4-羟基-3-亚硝基苯甲酰胺生物合成的最终步骤是通过NspF进行的,NspF是一种单加氧酶,可将邻氨基苯酚转化为相应的亚硝基产物(羟苯胺酶活性)。以前的NspF静止形式的生化特征表明该酶属于偶联双核铜酶(CBC)家族。该酶家族的另一个成员,酪氨酸酶,能够单加氧单酚(单酚酶活性),但不能使邻氨基酚。为了深入了解NspF的独特反应性,我们生成并表征了其活性位点的氧基形式。对与氧酪氨酸酶相同的光谱特征的观察表明,氧-NspF包含Cu_2O_2核心,其中过氧化物以μ-η〜2:η〜2模式配位,证实了NspF是CBC酶。发现该氧基形式与单酚反应,表明与酪氨酸酶一样,NspF也具有单酚酶活性。对单酚酶和羟基苯胺酶活性的两种亲电机理的比较表明,它们各自的过渡态之间存在较大的几何变化。在该酶家族具有等效的μ-η〜2:η〜2过氧桥联双核铜活性位点的差异反应性的背景下,讨论了每种过渡状态下蛋白质袋与底物之间发生特定相互作用的可能性。

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