The Structural Basis for Control of 5-Lipoxgenease Activity

描述（由申请人提供）：酶5-脂氧合酶（5-LOX）启动促炎白三烯的合成。这些脂质介质是由双分子层释放的花生四烯酸（AA）通过Ca2+依赖性磷脂酶A2的作用合成的。5-LOX活性是短暂的，时间控制部分是由于酶的内在不稳定性。这种不稳定性提供了一种自动调节机制，防止促炎白三烯的过度产生。然而，并非所有的脂氧合酶都存在“程序性淘汰”现象，目前已确定了稳定的同工型。我们建议解决控制5-LOX活性的三个关键方面：(1)产品特异性：5-LOX的底物是多不饱和类二十烷花生四烯酸。反应的第一步是从戊二烯的中心碳中提取氢。AA有3个戊二烯基团（和6个可能的过氧化位点，每个位点具有R-或S-手性）。然而，动物脂氧合酶通常产生单一的、区域和立体特异性的产物。我们将开发一个与其产品特异性一致的5-LOX特异性模型。我们有一个2.86E分辨率的工程5-LOX结构，为这些生化和结构研究奠定了基础。(2) 5-LOX的“程序性退化”似乎有两个组成部分：结构不稳定性和基于流失率的自杀抑制。我们的数据，包括我们稳定的5-LOX突变形式，表明5-LOX特有的特征导致了一个微妙的限制C-末端，这有助于5-LOX的不稳定性。提出了确定非周转率和基于周转率的失活分子基础的实验。(3)区室化Ca2+依赖的5-LOX膜结合将酶靶向底物库，并促进下游酶活性的接近。实验数据支持一个模型，其中特定的Ca2+结合位点稳定了5-LOX的c2样结构域的“插入环”。其他人认为5-LOX与其辅助蛋白FLAP（一种完整的膜蛋白）结合。我们建议通过实验来确定5-LOX与双分子层的相互作用，并确定催化结构域是否也与膜相互作用，以及FLAP是将底物交给酶，还是简单地将AA浓缩在膜上。

DESCRIPTION (provided by applicant): 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 propose to address three 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. We have a 2.86E resolution structure of an engineered 5-LOX that establishes the foundation for these biochemical and structural studies. (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. (3) Compartmentalization Ca2+- dependent membrane binding of 5-LOX targets the enzyme to substrate reservoirs and promotes proximity to downstream enzyme activities. Experimental data support a model in which specific Ca2+ binding sites stabilize "insertion loops" in the C2-like domain of 5-LOX. Others have suggested that 5-LOX binds to its helper protein FLAP, an integral membrane protein. We propose experiments to define the interaction of 5-LOX with the bilayer and determine whether the catalytic domain interacts with the membrane as well, and whether FLAP hands off the substrate to the enzyme, or simply concentrates the AA in the membrane. PUBLIC HEALTH RELEVANCE: Effective therapeutic strategies require that drugs be specific for their protein targets, and therefore the structures of these targets, as well as an understanding of their molecular mechanisms, are essential to guide the development of new medicines. Because of its pivotal role in the biosynthesis of inflammatory leukotrienes, the enzyme 5-lipoxygenase is a target for drugs to treat asthma. The proposed studies will provide both structural and mechanistic information for this key enzyme.