Determination of the Lid Residues Critical for Catalysis by Cholestrol Oxidase

一个有趣的界面催化问题是胆固醇氧化酶的例子。 这种水溶性酶将胆固醇从脂膜双层中提取出来，净运动约为10 A，进入深活性位点口袋。 底物被氧化和异构化，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位点。 X射线晶体结构的检查揭示了一个活性位点，其长度为11 A（适合于结合胆固醇），与FAD辅因子相邻，并通过两个表面环（5和20个残基长）与溶剂封闭。 (The脱氢异雄酮结合结构揭示了其中一个环中的1-2 A运动，以适应结合位点中的类固醇;胆固醇可能导致其C-17尾的更大重排。 据推测，一旦氧化酶扩散到脂质膜表面，这两个环必须打开以将疏水活性位点暴露于底物。 将解决的问题是：（1）氧化过程的机制是什么，它是酶促反应中的限速步骤？ (2)如果我们通过定点突变用丙氨酸取代表面环的侧链，酶螯合底物和中间体并催化它们转化的能力会发生什么变化？ 将研究丙氨酸取代突变对以下性质的影响：（i）催化速率和限速步骤，使用动力学同位素效应。 (ii)氧化过程，通过研究（a）脱氮FAD重建（B）环丙基自由基陷阱（c）诱变推定的总碱的影响。 (iii)对具有各种烷基C-17尾的羟基类固醇的亲和力和周转率。 所有这些实验将导致在二维脂质界面的结合和催化的模型。 该模型将与理解其他类固醇结合蛋白和酶的作用模式相关，例如，类固醇生物合成所需的酶和参与固醇转运的蛋白。 此外，胆固醇氧化酶在临床应用中广泛用于测定血清胆固醇水平。 了解结构如何影响催化作用将导致胆固醇氧化酶的设计更适合于固定化和临床测定目的。 作为拟议研究的结果，蛋白质中的机械构象变化如何影响结合和催化将得到更好的理解。 为特定目的改变和修饰酶功能的能力仍处于发展的婴儿阶段，一套创造结构和功能的一般规则才刚刚开始从广泛的观察中出现。 正是通过本提案中概述的详细研究，这些规则将变得更加明显。 %%% 动脉炎是一种在美国人群中广泛存在的疾病。 这种冠状动脉疾病的部分原因是血液中胆固醇含量过高。 降低这种疾病发病率的一种方法是监测个人的血液胆固醇水平，并为胆固醇水平不健康的人制定降低胆固醇水平的措施。 我们的研究将导致更容易和便宜的血液胆固醇水平测试的发展，这将导致更容易的预防措施处方。 这项研究的目的是研究蛋白质催化剂胆固醇氧化酶的结构与功能的关系。 根据现有的结构信息，尚不清楚底物胆固醇如何与催化剂结合或如何释放。 已经提出，催化剂的两个片段对于胆固醇的结合和释放是必需的。 我们将使用分子生物学技术来修饰这些片段的结构。 这些修饰影响胆固醇的结合和释放，使用血清胆固醇水平测定。 此外，这项研究将导致对类固醇（例如胆固醇）在体内转化和运输机制的基本了解。 从长远来看，有可能利用从这类研究中获得的信息开发新的生物材料。 蛋白质与脂质界面的相互作用还不清楚。 二维脂质界面可以构建与新的蛋白质连接到他们，这将允许胆固醇或其他类固醇的生物传感器的发展。 这些生物材料可能比目前可用的一些材料更稳定，更不易发生生物降解。 ***

Sampson 9405394 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 10A, 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 11A 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 A 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.) 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 questions that will be addressed are: (1) what is the mechanism of the oxidative process and is it the rate-limiting step in the enzymatic reaction? (2) if we substitute side-chains of the surface loops with alanine by site-directed mutagenesis, what happens to the ability of the enzyme to sequester substrates and intermediates and to catalyze their transformation? The consequences of the alanine-substitution mutations on the following properties will be investigated: (i) the catalytic rate and on the rate-limiting step, using kinetic isotope effects. (ii) the oxidative process, by studying the effects of (a) deazaFAD reconstitution (b) cyclopropyl radical traps (c) mutagenizing the putative general base. (iii) the affinity for and the turnover of hydroxysteroids with various alkyl C-17 tails. 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 effects 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. %%% Arteriosclerosis is a widespread disease amongst the American population. This coronary disease is partly a result of high levels of cholesterol in the blood. One way to reduce the incidence of this disease is to monitor the blood cholesterol levels of individuals and to prescribe measures to reduce cholesterol levels for those with unhealthy levels of cholesterol. Our research will lead to the development of more facile and inexpensive tests of blood cholesterol levels, that will lead to easier prescription of preventative measures. The goal of the proposed research is to study how the structure of the protein catalyst, cholesterol oxidase, relates to function. From the structural information that is available, it is not clear how the substrate, cholesterol, binds to the catalyst or how it is released. It has been proposed that two fragments of the catalyst are essential for binding and release of cholesterol. We will modify the structure of t hese fragments using molecular biological techniques. The modifications affect binding and release of cholesterol, use assays of serum cholesterol levels. In addition, this study will lead to basic insight into the mechanisms of steroid (for example, cholesterol) transformation and transport in the body. It may be possible in the long term to develop new biomaterials using information obtained from this type of study. The interaction of the protein with the lipid interface is not well understood. Two dimensional lipid interfaces may be constructed with novel proteins attached to them which will allow the development of biosensors for cholesterol or other steroids. These biomaterials may be more stable and less susceptible to biological degradation than some materials currently available. ***