BIOLOGICAL OXIDATION MECHANISMS

生物氧化机制

• 批准号: 6018279
• 负责人: VINCENT MASSEY
• 金额: $ 54.9万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018279
• 关键词: Escherichia coli; NAD(P)H dehydrogenase; active sites; aminoacid oxidase; chemical kinetics; circular dichroism; cofactor; enzyme mechanism; enzyme model; enzyme reconstitution; enzyme structure; flavins; flavoproteins; fluorescence spectrometry; isozymes; lactate dehydrogenases; molecular cloning; oxidoreductase; oxygenases; protonation; site directed mutagenesis; stop flow technique; synthetic enzyme

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.

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和 在生物系统中控制。 方法的方法将是 代表各类的特定酶的研究 黄素蛋白和模型系统的研究。 大量使用将与稳态动力学相结合 快速反应动力学研究以及取代的技术 黄素蛋白与合成黄素的天然共酶，以便为了 测试可能的机制，并探测环境的性质 立即在蛋白质结合的黄素周围。 后者特别是 在提供有关活动现场区域的信息中很重要 X射线无法获得结构的蛋白质 晶体学。 它还提供了一种调查的强大方法 在酶的情况下，蛋白质结构的动态方面 晶体结构是已知的。 我们还广泛使用 分子遗传学技术在阐明化学反应中 机制。 我们已经克隆并表达了L乳酸的基因 单加氧酶，MHPC氧化酶和旧黄色酶，正在携带 关于这些酶的位置定向突变体形式的详细研究，以及 L-乳酸氧化酶，D-氨基酸氧化酶，DT-磷酸盐酶，烷基的 氢过氧化物还原酶和P-羟基苯甲酸酯羟化酶。 全部 这些酶已经可以使用三维结构，或者 结构确定正在进行中。