CHOLESTEROL OXIDASE LOOP RESIDUES AND CATALYSIS

胆固醇氧化酶环残基和催化

• 批准号: 2029238
• 负责人: NICOLE S SAMPSON
• 金额: $ 9.54万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2029238
• 关键词: active sites; bacterial proteins; binding proteins; cholesterol; enzyme activity; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate analog; immobilized enzymes; lipid bilayer membrane; liposomes; oxidoreductase; protein structure function; site directed mutagenesis

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.

界面催化的一个有趣的问题是由 胆固醇氧化酶这种水溶性酶提取胆固醇 从脂膜双层中出来，净运动约为10 愤怒，进入一个很深的活动部位口袋。基底被氧化， 异构化，并且所得酮返回到脂质双层。 然而，没有明显的途径使底物到达 活性部位对X射线晶体结构的检查显示， 长度为11埃的位点（适合于结合胆固醇）， 与FAD辅因子相邻，并通过两个表面与溶剂隔离 环（5和20个残基长）。(The脱氢异雄酮结合 结构揭示了1-2埃的运动在一个环， 在结合位点容纳类固醇;胆固醇可能会导致 一个更大的重组与它的C-17尾巴。[2，3]假设： 这两个环必须打开以使疏水活性位点暴露于 一旦氧化酶扩散到脂质膜表面，底物就被氧化。的 这项研究的目的是确定哪些结构要素 是运动、结合和催化发生所必需的。 一 定点诱变研究和机制的组合 使用底物类似物的实验将解决表面环的作用 在粘合基材和产品中。研究以确定相对 酶结合物种的稳定性有望识别 中间体稳定所必需的酶结构。 此外，含有底物的脂质体的构建和底物 类似物允许在膜界面处的结合现象， 研究了所有这些实验将导致一个模型， 和在二维脂质界面的催化作用。这一模式将 与了解其他类固醇结合的作用方式有关 蛋白质和酶，例如， 类固醇和参与固醇运输的蛋白质的生物合成。 此外，胆固醇氧化酶广泛用于临床 用于测定血清胆固醇水平的应用。 了解结构如何影响催化作用将导致 设计更适合于固定化的胆固醇氧化酶， 临床分析目的。蛋白质的机械构象变化 结合和催化作用的效果将更好地理解， 提议的研究。改变和修饰酶功能的能力 一个具体的目的仍然处于发展的婴儿阶段， 创造结构和功能的一般规则才刚刚开始 从广泛的观察中得出的结论。怀着 本提案中概述的详细研究类型，这些规则将 变得更加明显。