Biological Oxidation Mechanisms

描述（由申请人提供）：该研究项目的目的是详细了解黄素辅酶的化学反应性，以及它们的化学多功能性如何在生物系统中被引导和控制。方法将是研究代表大多数主要类别的黄素蛋白的特定酶和研究游离黄素的模型系统。将大量使用稳态动力学与快速反应动力学研究相结合的方法，以及用合成黄素代替黄素蛋白的天然辅酶的技术，以测试可能的机制，并探测蛋白结合黄素周围环境的性质。这种技术不仅在提供关于蛋白质中活性位点区域的信息方面特别重要，其中结构不能从X射线晶体学中获得，而且还提供了一种强有力的方法，用于在已知晶体结构的酶的情况下研究蛋白质结构的动态方面。我们还广泛使用分子遗传学技术来阐明化学反应机制。我们已经克隆并表达了L-乳酸单加氧酶和老黄酶的基因，并通过我们的合作者获得了L-乳酸氧化酶、烷基氢过氧化物还原酶和theioredoxin还原酶的遗传修饰形式。对于所有这些酶，三维结构已经可用，或者结构确定正在进行中。

DESCRIPTION (provided by applicant): The aim of the research project is a detailed understanding of the chemical reactivity of the flavin coenzymes, and how their chemical versatility is channeled and controlled in biological systems. The methods of approach will be the study of specific enzymes representative of most of the main classes of flavoproteins and studies of model systems with free flavins. 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 fiavoproteins with synthetic flavins, in order to test possible mechanisms, and to probe the nature of the environment immediately around the protein-bound flavin. This technique is not only particularly important in providing information about the active site region in proteins where structures are not available from x-ray crystallography, but in addition 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 and Old Yellow Enzyme, and through our collaborators have access to genetically modified forms of L-lactate oxidase, alkyl hydroperoxide reductase and theioredoxin reductases. For all of these enzymes, three-dimensional structures are already available, or the structural determination is in progress.