FREE RADICALS IN BIOLOGICAL REDOX REACTIONS

生物环境中的自由基通常会引起破坏性的 由于它们的高反应性和非特异性化学反应。 然而，最近已经鉴定了一类酶，其中 高氧化性的氨基酸侧链自由基在水解过程中产生， 正常的催化过程。这些物种似乎是不可分割的 这些酶的正常功能。 了解这些自由基产生的方式， 稳定并致力于有效催化是一项重大的、长期的 项目的目标。 不成对电子- 自旋密度分布在径向中心上是一种手段， 其自由基反应性似乎受到控制。当地的蛋白质 氧化还原活性侧链周围的电荷是第二重要的 在引导这些物种的反应性的因素，以及程度， 它们被整个蛋白质结构所屏蔽， 三分之一 这些控制功能将在即将到来的 授予期内多种自由基产生和利用酶。 我们打算重点关注的具体系统包括 光系统I和II在放氧生物体中， 核糖核苷酸还原酶，胰高血糖素-H合酶，半乳糖氧化酶， 和光解酶。 其中一些系统的工作将专门由 在密歇根州;其他人将合作研究。 基团 由这些酶产生的包括酪氨酸、色氨酸， 组氨酸。 我们将应用的方法包括以下技术： 微生物学、分子生物学、生物化学和磁共振 谱 自由基侧链的选择性同位素标记 在激进分子的局部环境中， 无论是通过现有的技术，还是通过我们所使用的方法， 建议发展。 EPR和ENDOR光谱提供了主要的 分析工具，旨在评估未配对的电子自旋 密度分布和局部环境。 模型化合物和计算方法将用于补充 体内研究。 酪氨酸自由基是迄今为止最好的特征 并将继续是拟议工作的主要重点。 的 邻位取代的苯酚的影响，似乎发生在 半乳糖氧化酶，将进行研究。 到目前为止，含有自由基的 酶的发现有些偶然。 通过表征 这些物种在这些酶中的详细信息，它们是已知的， 发生，我们预计，其催化活性的一般原则， 将出现并导致它们在其他酶中的识别 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.