MECHANISMS OF ENZYMES WHICH FORM CARBON CARBON BONDS

形成碳碳键的酶的机制

• 批准号: 6498869
• 负责人: LINDA C. KURZ
• 金额: $ 18.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498869
• 关键词: Archaea; Raman spectrometry; bacterial proteins; citrate synthase; conformation; enolate; enzyme mechanism; histidine; infrared spectrometry; interferometry; molecular dynamics; nuclear magnetic resonance spectroscopy; thioester; Archaea; Raman spectrometry; bacterial proteins; citrate synthase; conformation; enolate; enzyme mechanism; histidine; infrared spectrometry; interferometry; molecular dynamics; nuclear magnetic resonance spectroscopy; thioester

DESCRIPTION: (Verbatim from the Applicant's Abstract) Our long-term goal is to understand the mechanisms of enzymes that catalyze carbon-carbon bond formation via Claisen condensations. Such enzymes are prominent in biosynthetic pathways (e.g., fats and cholesterol) and in energy-yielding pathways (e.g., tricarboxylic acid and glyoxylate cycles); we concentrate on citrate synthases. Their study will advance our general understanding of enzymatic catalysis. Citrate synthases are prototypes for several important catalytic strategies: carbonyl polarization to increase reactivity of the electrophilic substrate, facilitation of formation of the nucleophilic substrate carbanion, the use of unusual ionization states of histidine residues as acid catalysts, and changes in macromolecular conformation. We have four specific goals. 1. To complete determination of the detailed reaction profile using intramolecular substrate-isotope effects and transient kinetic methods (stopped-flow fluorescence and chemical quench). Oxygen exchange will be used to detect intermediates in the hydrolysis reaction. 2. To determine the structural basis of carbonyl polarization and carbanion stabilization. Structural and computational studies have implicated previously unstudied residues. We will change these residues to ones that cannot function in the ways propose and determine the consequences for carbonyl polarization (NMR and FTIR studies) and proton transfer (exchange studies). These solution results will be correlated with X-ray structures and calculations of the effects of mutants by our collaborators. 3. To determine the ionization states of the catalytic histidines in the various complexes of the enzyme. This exceeding important (but difficult) problem will be attacked using three different methodologies, solid-state NMR, solution-state NMR, and Raman spectroscopy. 4. To investigate the catalytic role of protein dynamics and flexibility by detailed mechanistic comparisons in a series of structurally homologous citrate synthases originating in organisms optimized to function at widely different temperatures. We shall correlate the stabilities of the intermediates and transition states with temperature to reveal which elementary steps in the mechanism are modulated by protein flexibility and dynamics. We will test predictions based on previous experimental and theoretical work.

描述：（逐字研究申请人的摘要）我们的长期目标是 了解催化碳碳键的酶的机制 通过Claisen冷凝。这种酶在生物合成途径中很突出 （例如，脂肪和胆固醇）和能量赋予能量的途径（例如， 三羧酸和乙二醇循环）；我们专注于柠檬酸盐合酶。 他们的研究将促进我们对酶促催化的一般理解。 柠檬酸盐合酶是几种重要催化策略的原型： 羰基极化以增加亲电底物的反应性， 促进亲核底物carbanion的形成，使用 组氨酸残基作为酸催化剂的异常电离状态，并且变化 在大分子构象中。我们有四个具体的目标。 1。完成 使用分子内确定详细的反应曲线 底物 - 同位素效应和瞬态动力学方法（停止流量 荧光和化学淬灭）。氧交换将用于检测 水解反应中的中间体。 2。确定结构基础 羰基两极化和碳纤维稳定。结构和 计算研究已暗示了先前未研究的残基。我们将 将这些残留物更改为无法以建议的方式起作用的残留物 确定羰基极化的后果（NMR和FTIR研究）和 质子转移（交换研究）。这些解决方案结果将相关 通过X射线结构和我们的突变体影响的计算 合作者。 3。确定催化的电离状态 酶的各种复合物中的组织氨酸。这超出了重要的 （但困难）问题将使用三种不同的方法攻击， 固态NMR，溶液状态NMR和拉曼光谱法。 4。调查 蛋白质动力学和灵活性的催化作用通过详细的机理 在一系列结构同源的柠檬酸合酶中进行比较 起源于优化可在广泛不同的生物体 温度。我们应将中间体的稳定性与 过渡状态具有温度，以揭示哪些基本步骤 机制受蛋白质的柔韧性和动力学调节。我们将测试 基于先前的实验和理论工作的预测。