FREE RADICALS IN BIOLOGICAL REDOX REACTIONS

生物环境中的自由基通常会引发破坏性的 由于它们的高反应性和非特异性化学而导致的反应。 然而，最近发现了一类酶，在这种酶中 高氧化性氨基酸侧链自由基在反应中产生 正常的催化过程。这些物种似乎是 这些酶的正常功能。 了解这些自由基是如何产生的， 稳定，并针对有效的催化是一个重大的、长期的 项目的目标。未配对的电子-- 自旋密度分布在自由基中心上是一种方式 其激进的反应性似乎得到了控制。当地的蛋白质 氧化还原活性侧链周围的环境是第二个重要因素 引导这些物种的反应性的因素，以及 它们被整体的蛋白质结构保护而不被溶解 第三个。这些控制特征将在接下来的 在各种自由基产生和利用酶的授权期。 我们打算重点关注的具体系统包括 下面是：放氧生物中的光系统I和II， 核苷酸还原酶，前列腺素-H合成酶，半乳糖氧化酶， 和光解酶。其中一些系统的工作将专门完成 在密歇根州立大学；其他人将进行合作研究。激进分子 由这些酶产生的物质包括酪氨酸、色氨酸，可能还有 组氨酸。我们将应用的方法包括 微生物学、分子生物学、生物化学和磁共振 光谱学。自由基侧链的选择性同位素标记 可能在当地环境中的激进分子将是 无论是通过现有技术还是通过我们 建议发展。EPR和Endor光谱仪提供了主要的 分析工具，旨在评估未配对的电子自旋 密度分布和自由基部位周围的局部环境。 将使用模型化合物和计算方法来补充 活体研究。到目前为止，酪氨酸自由基是最好的表征 并将继续是拟议工作的主要重点。这个 苯酚的邻位取代的影响，似乎发生在 半乳糖氧化酶，将被研究。到目前为止，含有自由基的 酶在某种程度上是偶然发现的。通过刻画 这些物种在已知它们所在的那些酶中有详细的 发生时，我们预计它们催化活性一般原理 会出现并导致它们在其他酶中被鉴定 反应。

Free radicals in biological milieu generally provoke destructive reactions owing to their high reactivity and non-specific chemistry. Recently, however, a class of enzymes has been identified in which highly oxidizing amino acid side chain radicals are generated during the normal course of catalysis. These species appear to be integral to the proper function of these enzymes. Understanding the means by which these radicals are generated, stabilized, and directed to effective catalysis is a major, long-term objective of the project. The manner in which the unpaired electron- spin density is distributed over the radical center is one means by which radical reactivity appears to be controlled. The local protein environnent around the redox active side chain is a second important factor in channeling the reactivity of these species, and the extent to which they are shielded from sovent by the overall protein structure is a third. These controlling features will be studied in the upcoming grant period in a variety of radical generating and utilizing enzymes. The specific systems upon which we intend to focus include the following: Photosystem I and II in oxygen-evolving organisms, ribonucleotide reductase, prostaglandin-H synthase, galactose oxidase, and photolyase. Work on some of these systems will be done exclusively at Michigan State; others will be studied collaboratively. The radicals generated by these enzymes include tyrosine, tryptophan, and possibly histidine. The methods we will apply include techniques from microbiology, molecular biology, biochemistry, and magnetic resonance spectroscopy. Selective isotopic labelling of the radical side chains potentially in the local environment of the radical will be accomplished, either by existing techniques or by methods that we propose to develop. EPR and ENDOR spectroscopies provide the primary analytical tools and are intended to assess the unpaired electron-spin density distribution and the local environment around the radical site. Model compounds and computational methods will be used to complement the in vivo studies. Tyrosine radicals are the best characterized thus far and will continue to be the primary focus of the proposed work. The effects of ortho substitution of the phenol, as appears to occur in galactose oxidase, will be studied. Thus far, radical-containing enzymes have been uncoverd somewhat accidentally. By characterizing these species in detail in those enzymes in which they are known to occur, we anticipate that general principles of their catalytic activity will emerge and lead to their identification in other enzymatic reactions.