Mechanistic Studies of Tryptophan Oxidizing Enzymes

色氨酸氧化酶的机理研究

• 批准号: 0843537
• 负责人: Aimin Liu
• 金额: $ 57.72万
• 依托单位: Georgia State University Research Foundation, Inc.
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843537&HistoricalAwards=false
• 关键词: Mechanistic; Studies; Tryptophan; Oxidizing; Enzymes

Heme iron active sites are present in a wide variety of proteins and enzymes. The different biological functions they perform include oxygen binding, storage, transfer, and activation for hydroxylation, peroxidation, and desaturation. This project examines the molecular biochemistry exhibited by two structurally diverse heme-containing enzymes that oxidize either free tryptophan or protein-bound tryptophan residues. Tryptophan 2,3-dioxygenase (TDO) inserts two oxygen atoms into free tryptophan in a four electron oxidizing process by a b-type heme cofactor. This enzyme represents a potentially new hemoprotein dioxygenase superfamily whose oxygenase activity remains poorly understood. MauG is a novel enzyme that utilizes two c-type hemes to catalyze a posttranslational modification of a 119 KDa protein. Such a modification endows endogenous tryptophan residues with a new catalytic activity. The MauG-catalyzed reaction is a six-electron oxidation process and the utilization of two c-type hemes to perform a hydroxylation reaction and the subsequent oxidation reactions are unprecedented. Novel high valent iron intermediates have been trapped from these tryptophan oxidizing enzymes. This research will establish a better definition of the requirements needed to attain the unusual Fe(IV) intermediates as well as elucidate their relation to oxidizing the free or protein-bound tryptophan substrates. A biochemical and spectroscopic approach employing site-directed mutagenesis, electron paramagnetic resonance (EPR) spectroscopy, Mössbauer spectroscopy, mass spectrometric analysis, and structural determination as well as quantum mechanics computational methods will be utilized to study the electronic structure of the intermediates in these enzymes. These studies are directed toward obtaining detailed insight into electronic and geometric structural contributions to the formation and stabilization of high valent iron intermediates, the electronic structure of reactive oxygen intermediates, and the heme-dependent tryptophan oxidizing mechanisms contrasts to non-heme catalysis. Broader Impacts: This research will provide the opportunity for motivated students, including underrepresented populations, to participate in the frontier of molecular biochemistry research. Students exposure to an array of biochemical and biophysical techniques will be integrated with hands-on training. A video tutorial of electron paramagnetic resonance (EPR) spectroscopy will be developed during the experimental process to provide a thorough demonstration of EPR-centered techniques in studying the chemical properties of important biomolecular systems.

血红素铁活性位点存在于多种蛋白质和酶中。它们的不同生物学功能包括氧结合、储存、转移和活化羟基化、过氧化和去饱和。该项目研究了两种结构不同的含血红素酶所表现出的分子生物化学，这些酶氧化游离色氨酸或蛋白质结合色氨酸残基。色氨酸2,3-双加氧酶（TDO）通过b型血红素辅助因子在四电子氧化过程中将两个氧原子插入游离色氨酸中。这种酶代表了一种潜在的新的血红蛋白双加氧酶超家族，其加氧酶活性仍然知之甚少。MauG是一种利用两个c型血红素催化119 KDa蛋白翻译后修饰的新型酶。这种修饰使内源性色氨酸残基具有新的催化活性。mag催化的反应是一个六电子氧化过程，利用两个c型血红素进行羟基化反应和随后的氧化反应是前所未有的。从这些色氨酸氧化酶中捕获了新的高价铁中间体。这项研究将更好地定义获得不寻常的铁（IV）中间体所需的条件，并阐明它们与氧化游离或蛋白质结合色氨酸底物的关系。利用位点诱变、电子顺磁共振（EPR）光谱、Mössbauer光谱、质谱分析、结构测定以及量子力学计算方法等生化和光谱方法，将研究这些酶中间体的电子结构。这些研究旨在详细了解电子和几何结构对高价铁中间体的形成和稳定的贡献，活性氧中间体的电子结构，以及与非血红素催化相比，血红素依赖的色氨酸氧化机制。更广泛的影响：这项研究将为有动力的学生提供机会，包括代表性不足的人群，参与分子生物化学研究的前沿。学生接触一系列生化和生物物理技术将与实践训练相结合。在实验过程中，将开发电子顺磁共振（EPR）光谱的视频教程，以全面演示以电子顺磁共振为中心的技术在研究重要生物分子体系的化学性质方面的应用。