FUNCTIONAL STUDIES OF HUMAN AND SOYBEAN LIPOXYGENASE

人类和大豆脂氧合酶的功能研究

• 批准号: 2910348
• 负责人: Theodore R Holman
• 金额: $ 9.85万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910348
• 关键词: Mossbauer spectrometry; X ray spectrometry; active sites; chemical kinetics; chemical structure function; circular dichroism; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; iron; lipoxygenase; mutant; site directed mutagenesis; soybeans

DESCRIPTION: Lipoxygenases (LOs) are enzymes that catalyze the incorporation of dioxygen into 1,4-cis,cis-pentadiene containing fatty acids, and are widely distributed throughout the plant and animal kingdoms. The activity of the enzyme has been implicated in plant germination, human asthma and arthritis. The active site of the enzyme contains an essential iron atom. Two distinct mechanisms for LO catalysis have been proposed. The first involves a radical based mechanism in which the active site ferric ion oxidizes the 1,4-diene of linoleic acid (LA) to a fatty acid radical. The second mechanism uses the ferric iron to facilitate de-protonation of the substrate by coordinating the resulting carbonation to form an organometallic intermediate. Thus, the key difference in the two mechanisms is the role of the iron center and how it activates the proton abstraction. This proposal involves spectroscopic and kinetic studies aimed at investigating the structure and function of the active site iron in both soybean and human lipoxygenases. In addition, a number of active site mutants designed to affect the electronic structure of the iron and its reactivity will be studied. Spectroscopic techniques (e.g., EPR, MCD, EXAFS) will be used to determine the changes in the iron coordination and/or electronic environment. These studies will be correlated to kinetic investigations that address the effect of the structural changes on reactivity as well as the effects of pH, viscosity and isotopic substitutions. The information obtained will be used to determine a unified theory of the structure and function of the Fe center in catalyzing the oxidation reactions.

描述：脂氧合酶（LO）是催化 将分子氧掺入含脂肪酸的1，4-顺式、顺式-戊二烯中 酸，并广泛分布于整个植物和动物王国。 这种酶的活性与植物的发芽、人类的生长和发育有关。 哮喘和关节炎。 酶的活性部位含有一个必需的 铁原子 已经提出了两种不同的LO催化机制。 第一个涉及一个自由基为基础的机制，其中活性位点铁 离子将亚油酸（LA）的1，4-二烯氧化成脂肪酸自由基。 第二种机制使用三价铁来促进 通过协调所得的碳酸化作用， 有机金属中间体 因此，这两种机制的关键区别在于 是铁中心的作用以及它如何激活质子提取。 这项建议涉及光谱和动力学研究，目的是 研究了活性中心铁的结构和功能， 大豆和人脂氧合酶。 此外，一些活跃的网站 突变体的设计，以影响电子结构的铁及其 将研究反应性。 光谱技术（例如，EPR，MCD， EXAFS）将用于确定铁配位和/或 电子环境。 这些研究将与动力学相关 调查，解决结构变化对 反应性以及pH、粘度和同位素的影响 替代品 所获得的信息将用于确定一个统一的理论， Fe中心的结构与催化氧化作用 反应.