STRUCTURAL STUDIES ON LIPOXYGENASES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 5-脂氧合酶（5-LOX）启动促炎性白三烯的合成。这些脂质介质由花生四烯酸（AA）合成，花生四烯酸（AA）通过Ca 2+依赖性磷脂酶A2的作用从双层释放。5-LOX活性是短暂的，并且时间控制部分地由于酶的内在不稳定性而出现。这种不稳定性提供了一种自动调节机制，防止促炎性白三烯的过度产生。然而，“程序性陈旧”并不是所有的脂氧合酶所共有的，并且已经鉴定了稳定的同种型。我们提出了控制5-LOX活性的两个关键方面：（1）产物特异性5-LOX的底物是多不饱和花生四烯酸。反应的第一步是从戊二烯的中心碳中提取氢。AA具有三个戊二烯部分（和六个可能的过氧化位点，每个具有R-或S-手性）。然而，动物脂氧合酶通常产生单一的、区域特异性和立体特异性产物。我们将开发一种与其产品特异性一致的5-LOX特异性模型。(2)5-LOX中的“程序性过时”似乎有两个组成部分：结构不稳定性和基于转换的自杀抑制。我们的数据，包括我们的5-LOX的稳定突变体形式，表明5-LOX独特的特征导致了导致5-LOX不稳定的微弱限制的C-末端。实验，以确定非营业额和营业额为基础的失活的分子基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The enzyme 5-lipoxygenase (5-LOX) initiates the synthesis of pro-inflammatory leukotrienes. These lipid mediators are synthesized from arachidonic acid (AA) released from the bilayer by the action of Ca2+-dependent phospholipase A2. 5-LOX activity is short-lived, and temporal control appears in part due to an intrinsic instability of the enzyme. This instability provides a mechanism for auto-regulation, preventing an over-production of pro-inflammatory leukotrienes. However, "programmed obsolescence" is not common to all lipoxygenases, and stable isoforms have been identified. We address two critical aspects of control of 5-LOX activity: (1) Product specificity The substrate for 5-LOX is the polyunsaturated eicosanoid arachidonic acid. The first step of the reaction is the abstraction of hydrogen from the central carbon of a pentadiene. AA has three pentadiene moieties (and six possible sites of peroxidation, each with either R- or S- chirality). Yet animal lipoxygenases generally produce a single, regio- and stereo- specific product. We will develop a model for 5-LOX specificity that is consistent with its product specificty. (2) Programmed obsolescence "Programmed obsolescence" in 5-LOX appears to have two components: structural instability and turnover-based suicide inhibition. Our data, including our stable mutant form of 5-LOX, suggest that features unique to 5-LOX result in a tenuously restrained C-terminus that contributes to 5-LOX instability. Experiments to define the molecular basis for non-turnover and turnover-based inactivation are proposed.