DIRECT OBSERVATION OF ENZYMATIC CATALYTIC STRATEGIES

直接观察酶催化策略

• 批准号: 3283973
• 负责人: LINDA C. KURZ
• 金额: $ 6.63万
• 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 1988-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283973
• 关键词: acetyl coA acetyltransferase; adenosine deaminase; citrate synthase; 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. To do so, we must go beyond a specification of the number and structures of stable intermediates and their rates of interconversion to an understanding of the structures of transition states and the means by which the free energy cost for reaching them has been minimized. We shall investigate three proposed origins of enzyme catalytic enhancement; local destabilization of reacting bonds in substrates, stabilization of reactive intermediates and stabilization of transition states. We have chosen two enzymes, citrate synthase and adenosine deaminase, for special emphasis because sufficient structural and kinetic data are available to suggest which catalytic strategies are used by these enzymes, and because our preliminary experiments show that intermediate structures characteristic of those strategies can be directly observed. Citrate synthase, which catalyzes the first step in the energy yielding citric acid cycle must activate both the carbonyl of a keto acid and the methyl of an acetyl thioester. One or the other of these two events is also critical to the reactions catalyzed by other quite different enyzmes that are vital to the energy yielding reactions of glycolysis and to biosynthetic steps in the synthesis of fats and cholesterol. Adenosine deaminase, whose proper functioning is necessary to the integrity of the immune response, probably operates by stabilizing a reactive intermediate, a catalytic strategy used by several other important enzymes. We shall use techniques, NMR and FTIR spectroscopy, which are sensitive to local bond distortions and to the electronic environment and thus will allow direct observation of the molecular structures which reflect the catalytic strategies being used and provide some insight into the enzyme-ligand interactions which allow them to occur. We also shall make the kinetic and equilibrium measurements which are required for proper interpretation or deeper understandng of the spectroscopic data. We shall investigate selected other systems (including malate synthase, HMG-CoA synthase, thiolase(s), cytidine and guanine deaminases) 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 understanding of disease and how to combat it. The primary thrust of this proposal is toward the answers to one of the fundamental questions in current chemical biology.

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。 为此，我们必须超越数字的规范 和稳定中间体的结构及其互换率 了解过渡状态的结构和手段 到达它们的自由能量成本已被最小化。 我们将 研究酶催化增强的三个提出的起源。当地的 反应键在底物中的稳定，反应性稳定 过渡状态的中间和稳定。 我们选择了两个 酶，柠檬酸盐合酶和腺苷脱氨酶，以特殊重点 因为可以建议足够的结构和动力学数据 这些酶使用了哪些催化策略，因为我们 初步实验表明中间结构的特征 这些策略可以直接观察到。 柠檬酸合酶 催化能量产生柠檬酸周期的第一步 激活酮酸的羰基和乙酰基的甲基 锡氏。 这两个事件中的一个或另一个对 反应是由其他完全不同的Enyzmes催化的，对 能量产生糖酵解和对生物合成步骤的反应 脂肪和胆固醇的合成。 腺苷脱氨酶，其适当 功能对于免疫反应的完整性是必要的，可能 通过稳定反应性中间体的催化策略来运行 由其他一些重要的酶。 我们将使用NMR和FTIR技术 光谱法对局部键失真敏感，对 电子环境，因此将直接观察 反映所使用的催化策略的分子结构和 提供一些有关允许它们的酶 - 配体相互作用的见解 发生。 我们还将进行动力学和平衡测量 需要正确解释或更深入地了解 光谱数据。 我们将调查选定的其他系统（包括 苹果酸合酶，HMG-COA合酶，硫醇酶（S），胞苷和鸟嘌呤 脱氨酶）为了扩展我们可以评估一般的环境 我们结论的适用性。 对代谢的详细理解 最基本层面的过程（酶的工作原理）不禁不得不 导致对疾病以及如何对抗疾病有更好的了解。 这 该提议的主要目的是向其中一个的答案 当前化学生物学的基本问题。