BIOLOGICAL OXIDATION MECHANISMS

该研究项目的主要目的是了解化学物质 黄素辅酶的反应性，以及如何使用这种反应性， 控制在生物系统中。 接近的方法将是 研究代表各种类别的特定酶， 黄素蛋白和模型系统的研究。 将相当多地使用稳态动力学， 快速反应动力学研究，以及取代 天然辅酶的黄素蛋白与合成黄素，以 测试可能的机制，并探索环境的性质 紧挨着蛋白质结合的黄素。 后者特别 重要的是提供有关活性部位区域的信息， 不能从X射线中获得结构的蛋白质 结晶学 它还提供了一种强有力的方法来调查 在酶的情况下，蛋白质结构的动态方面， 晶体结构已知。 我们还广泛利用 化学反应解析中的分子遗传学技术 机制等 我们已经克隆并表达了L-乳酸的基因 单加氧酶、MHPC加氧酶和老黄酶，并携带 详细研究了这些酶的定点突变形式， L-乳酸氧化酶、D-氨基酸氧化酶、DT-心肌黄酶、烷基 氢过氧化物还原酶和对羟基苯甲酸羟化酶。 所有 这些酶的三维结构已经可用，或者 结构确定正在进行中。

The main aim of the research project is an understanding of the chemical reactivity of flavin coenzymes, and how this reactivity is used and controlled in biological systems. The methods of approach will be the study of specific enzymes representative of the various classes of flavoproteins and studies of model systems. Considerable use will be made of steady state kinetics coupled with rapid reaction kinetics studies, and of the technique of replacing the natural coenzymes of flavoproteins with synthetic flavins, in order to test possible mechanisms, and to probe the nature of the environment immediately around the protein-bound flavin. The latter is particularly important in providing information about the active site region in proteins where the structure is not available from X-ray crystallography. It also offers a powerful way for investigating dynamic aspects of protein structure in the case of enzymes where the crystal structure is known. We are also making extensive use of molecular genetics techniques in the elucidation of chemical reaction mechanisms. We have cloned and expressed the genes for L-lactate monooxygenase, MHPC oxygenase and Old Yellow Enzyme, and are carrying out detailed studies on site-directed mutant forms of these enzymes, and of L-lactate oxidase, D-amino acid oxidase, DT-diaphorase, alkyl hydroperoxide reductase and p-hydroxybenzoate hydroxylase. For all of these enzymes the three dimensional structure is already available, or the structural determination is in progress.