CHOLESTEROL OXIDASE LOOP RESIDUES AND CATALYSIS

界面催化的一个耐人寻味的问题以 胆固醇氧化酶。这种水溶酶能提取胆固醇。 走出脂膜双层，净运动约10% 上千磅，放进一个很深的主动站点口袋里。该衬底被氧化并 异构化，生成的酮返回到脂质双层。 然而，没有明显的途径让底物到达 活动站点。对X-射线晶体结构的检查显示， 11埃长的部位(适合结合胆固醇)， 与FAD辅因相邻，并通过两个表面与溶剂隔开 环(5个和20个残基长)。(脱氢异雄酮结合 结构显示在其中一个环路中有1-2埃的运动 在结合部位调节类固醇；据推测，胆固醇可能导致 带有C-17尾巴的更大的重排。 这两个环路必须打开才能将疏水活性部位暴露给 一旦酶扩散到脂膜表面，底物就会被氧化。这个 这项拟议研究的目标是确定哪些结构元素 是发生运动、结合和催化所必需的。一个 定点突变研究与机制研究的结合 使用底物模拟的实验将解决表面环的作用 结合底物和产物。研究以确定相对的 酶结合物种的稳定性有望鉴定其成分 中间稳定所必需的酶结构。 此外，含有底物和底物的脂质体的构建 类似物允许膜界面上的结合现象 调查过了。所有这些实验都将产生一个绑定模型 以及二维脂分子界面上的催化作用。这款车型将是 与理解其他类固醇结合的作用方式有关 蛋白质和酶，例如，蛋白质和酶 参与类固醇运输的类固醇和蛋白质的生物合成。 此外，胆固醇氧化酶在临床上有广泛的应用。 应用于血清胆固醇水平的测定。 了解结构如何影响催化将导致 一种更适合于固定化和非固定化的胆固醇氧化酶的设计 临床化验目的。蛋白质中的机械性构象变化 影响结合和催化的结果将被更好地理解 拟开展的研究。改变和修改酶功能的能力 一个特定的目标仍处于发展的婴儿阶段，而一套 创建结构和功能的一般规则才刚刚开始 从已经进行的广泛观察中脱颖而出。它是与 本提案中概述的详细研究类型，这些规则将 变得更加明显。

An intriguing problem of interfacial catalysis is exemplified by the enzyme cholesterol oxidase. This water soluble enzyme extracts cholesterol out of the lipid membrane bilayer, with a net movement of approximately 10 Angstroms, into a deep active site pocket. The substrate is oxidized and isomerized, and the resulting ketone is returned to the lipid bilayer. There is no obvious pathway, however, for the substrate to reach the active site. Examination of the X-ray crystal structure reveals an active site that is 11 Angstroms long (suitable for binding cholesterol), adjacent to the FAD cofactor, and closed off from solvent by two surface loops (5 and 20 residues long). (The dehydroisoandrosterone bound structure reveals a 1-2 Angstrom movement in one of the loops to accommodate the steroid in the binding site; cholesterol presumably causes a larger rearrangement with its C-17 tail.[2, 3]) It is postulated that these two loops must open to expose the hydrophobic active site to the substrate once the oxidase has diffused to the lipid membrane surface. The goal of this proposed research is to determine what structural elements are necessary for movement, binding, and catalysis to occur. A combination of site-directed mutagenesis studies and mechanistic experiments using substrate analogs will address the role of surface loops in binding substrate and product. Studies to determine the relative stabilities of enzyme-bound species promise to identify the components of the enzyme structure necessary for intermediate stabilization. Furthermore, construction of liposomes containing substrate and substrate analogs allows binding phenomena at the membrane interface to be investigated. All of these experiments will lead to a model for binding and catalysis at the two-dimensional lipid interface. This model will be relevant to understanding the mode of action of other steroid binding proteins and enzymes, for example, the enzymes required for the biosynthesis of steroids and proteins involved in sterol transport. Furthermore-, cholesterol oxidase is used extensively in clinical applications for the determination of serum cholesterol levels. Understanding how the structure effects catalysis will result in the design of a cholesterol oxidase more suitable for immobilization and clinical assay purposes. How mechanical conformational changes in proteins effect binding and catalysis will be better understood as a result of the proposed research. The capability to alter and modify enzyme function for a specific purpose is still in the infant phases of development, and a set of general rules for creating structure and function is only beginning to emerge from the wide range of observations that have been made. It is with the type of detailed study outlined in this proposal that these rules will become more apparent.