DIRECT OBSERVATION OF ENZYMATIC CATALYTIC STRATEGIES

直接观察酶催化策略

• 批准号: 3283977
• 负责人: LINDA C. KURZ
• 金额: $ 9.41万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-27 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283977
• 关键词: acetyl coA acetyltransferase; adenosine deaminase; citrate synthase; enzyme mechanism; enzyme structure; enzyme substrate; fluorescence spectrometry; infrared spectrometry; nuclear magnetic resonance spectroscopy; thermodynamics

The long term goal of the proposed research is to understand at the molecular level how enzymes achieve their extraordinary catalytic efficiencies. We seek this understanding through direct observation of ligands (substrates, intermediates, and ground-state and transition-state analog inhibitors) bound to enzyme active sites using techniques (primarily NMR and FTIR spectroscopy) sensitive to local bond distortions, electronic environment, and other factors likely to reflect the enzyme's catalytic strategy. We will study the effects of structural alterations in substrates or their analogs and in the enzyme itself. Structural changes in the enzyme will be made using newly developed protein engineering methodology. In addition we shall make whatever kinetic and equilibrium measurements are required for proper interpretation or deeper understanding of the spectroscopic data. We have chosen two groups of enzymes, the Claisen enzymes and the nucleoside aminohydrolases for special emphasis because efficient structural and kinetic data are available to suggest what catalytic strategies be used by these enzymes and our past work shows that intermediate structures characteristic of those strategies can be directly observed. Enzymes which catalyze Claisen condensations are prominent in pathways requiring carbon-carbon bond formation in he biosynthesis of fats and cholesterol (thiolase(s), HMG-CoA synthase) as well in the energy-yielding pathways of glycolysis (citrate synthase) and the malate-isocitrate cycle malate synthase). These enzymes operate through activation of a substrate carbonyl together with stabilization of the alpha-carbanion of the acylthioester cosubstrate. Enzymes which catalyze amino- nucleoside(tide) hydrolysis have more varied but no less vital oles. Adenosine deaminase is necessary to the integrity of the immune response. Other enzymes of this class participate in salvage pathways of purines and pyrimidines guanase, etc) or are involved in the maintenance of cellular energy balance (adenylic deaminase). These enzymes operate through stabilization of tetrahedral intermediate, an analytic strategy used by several other important enzyme groups. Our work will first concentrate on citrate synthase and adenosine deaminase since a great deal of progress as already been made in understanding these enzymes and then our focus will shift to elected other systems within the two groups to expand the context within which we may assess the general applicability of our conclusions. A detailed understanding of metabolic processes at the most fundamental level (how enzymes work) cannot help but lead to a better understand of disease and how to combat it.

拟议研究的长期目标是在 分子水平酶如何实现其非凡催化 效率。 我们通过直接寻求这种理解 观察配体（底物，中间体和地面 与活性酶结合的过渡状态类似物抑制剂） 使用技术（主要是NMR和FTIR光谱法）的站点 对局部键扭曲，电子环境和 其他可能反映酶的催化策略的因素。 我们将研究底物结构改变的影响 或它们的类似物和酶本身。 结构性变化 该酶将使用新开发的蛋白质工程制成 方法论。 此外，我们将做任何动力学和 正确解释需要平衡测量或 对光谱数据的更深入了解。 我们选择了两个 一组酶，claisen酶和核苷 特别重点的氨基氢酶，因为有效的结构 并且动力学数据可以提出哪些催化策略 这些酶和我们过去的工作显示出中间的作品 这些策略的结构可以直接 观察到。 催化Claisen冷凝的酶是 在需要碳碳键形成的途径中突出 他的脂肪和胆固醇生物合成（硫代酶（S），HMG-COA 合成酶）以及糖酵解能量的途径 （柠檬酸酸酯合酶）和苹果酸 - 异构酸周期苹果酸 合酶）。 这些酶通过激活底物运行 羰基与稳定的Alpha-Carbanion 酰基三木蛋白酶覆盖物。 催化氨基的酶 核苷（潮汐）水解的多种多样，但重要 橄榄。 腺苷脱氨酶对于完整性是必要的 免疫反应。 该类的其他酶也参与 嘌呤和嘧啶鸟酶等的打捞途径等）或 参与细胞能量平衡的维持 脱氨酶）。 这些酶通过稳定 四面体中间体，几种分析策略 其他重要的酶组。 我们的工作将首先集中 柠檬酸合酶和腺苷脱氨酶，因为很多 在理解这些酶和 然后，我们的重点将转移到两者中当选的其他系统 扩大我们可以评估的背景的群体 我们结论的一般适用性。 详细的理解 最基本水平的代谢过程（酶如何 工作）不禁会更好地理解疾病和 如何对抗它。