Reactive Intermediates in Lipoxygenase Pathways

脂氧合酶途径中的反应中间体

• 批准号: 6616508
• 负责人: BETTY J GAFFNEY
• 金额: $ 20.55万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-10 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6616508
• 关键词: binding sites; biological signal transduction; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; eukaryote; free radicals; heme oxygenase; hydrogen peroxide; intermolecular interaction; iron; linoleate; lipoxygenase; manganese; oxidation; oxidation reduction reaction; unsaturated fatty acids

DESCRIPTION (provided by applicant): The overall goal of the studies proposed is to develop detailed understanding of how the enzymes interacting with hydroperoxides of polyunsaturated fatty acids (PUFA) cause electron redistribution in their free radical intermediates. In eukaryotic cells, hydroperoxides of polyunsaturated fatty acids (PUFA) are formed and rapidly converted to signaling molecules in dynamic complexes of several enzymes. The balance of products resulting from separate PUFA oxidation pathways is a subject critical to current pharmaceutical treatment of inflammatory and cardiovascular disorders. This proposal focuses on the mechanisms of electron rearrangements in two representative enzymes forming, and acting on, PUFA hydroperoxides. The specific enzymes chosen for study include manganese lipoxygenases (MnLO and mutants of iron lipoxygenase), that form PUFA hydroperoxides and an animal allene oxide synthase (AOS), that converts PUFA hydroperoxides to downstream signals. Each of the enzymes is chosen because of novel catalytic features. The specific aims are: (1) To examine, by high frequency electron paramagnetic resonance (hfEPR), activation of MnLO by two of its products, 11S- and 13R-hydroperoxides of linoleic acid. (2) To compare reduction potentials of manganese and iron in lipoxygenases using hfEPR. (3) To examine hfEPR spectroscopy and reduction potential of iron lipoxygenase mutated to include sequences that specify manganese binding. (4) To characterize functions and oxidation kinetics of AOS mutated at native tyrosine sites. (5) To obtain very high frequency EPR (vhfEPR) data on the microenvironment of the AOS tyrosine radical. The sensitivity of high frequency EPR (94 GHz), and simulation of EPR spectra, will facilitate many of these studies.

描述（由申请人提供）：提出的研究的总体目标是详细了解与多不饱和脂肪酸（PUFA）的氢过氧化物如何在其自由基中间体中引起电子重新分布。 在真核细胞中，形成多不饱和脂肪酸（PUFA）的水自生物氧化，并迅速转化为几种酶的动态复合物中的信号分子。单独的PUFA氧化途径产生的产品平衡是对当前炎症和心血管疾病的药物治疗至关重要的主题。 该提案重点介绍了两个代表性酶形成的电子重排的机制，并作用于PUFA氢过氧化物。选择用于研究的特异性酶包括形成PUFA氢过氧化物的锰（MNLO和铁氧化酶的突变体）和将PUFA氢氧化物转化为下游信号的PUFA氢过氧化物（AOS）。由于新型催化特征，选择了每种酶。具体目的是： （1）通过高频电子顺磁共振（HFEPR）检查MNLO的两种产品，即11s和13R亚氧化亚油酸的氧化物。 （2）比较使用HFEPR比较锰和铁的还原电位。 （3）检查HFEPR光谱和减少脂氧酶突变以包括指定锰结合的序列。 （4）表征在天然酪氨酸部位突变的AOS的功能和氧化动力学。 （5）获得有关AOS酪氨酸自由基微环境的非常高的EPR（VHFEPR）数据。 高频EPR（94 GHz）的敏感性以及EPR光谱的模拟将有助于许多此类研究。