Biological Oxidation Mechanisms

描述(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活性，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黄素蛋白的特定酶和研究含有游离黄素的模型系统。将大量使用稳态动力学和快速反应动力学研究，以及用合成黄素取代黄素蛋白的天然辅酶的技术，以测试可能的机制，并探索与蛋白质结合的黄素周围环境的性质。这项技术不仅在提供蛋白质中结构无法从X射线结晶学获得的活性部位区域的信息方面特别重要，而且还为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提供了一种强有力的方法。我们还广泛使用分子遗传学技术来阐明化学反应机理。我们克隆并表达了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转基因形式的L乳酸氧化酶、烷基过氧化氢还原酶和氧化还蛋白还原酶。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.