The Structural Basis for Control of 5-Lipoxgenease Activity

描述（由申请人提供）：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活性的三个关键方面：（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。我们有一个工程5-LOX的2.86E分辨率结构，为这些生化和结构研究奠定了基础。(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5-LOX不稳定性的微弱限制的C-末端。提出了确定非周转和基于周转的失活的分子基础的实验。(3)5-LOX的区室化Ca 2+依赖性膜结合将酶靶向于底物储库并促进接近下游酶活性。实验数据支持一个模型，其中特定的Ca 2+结合位点稳定的“插入环”的C2-样结构域的5-LOX。其他人认为5-LOX与其辅助蛋白FLAP结合，FLAP是一种完整的膜蛋白。我们建议实验来定义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是否将底物传递给酶，或者只是将AA浓缩在膜中。

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.