MECHANISMS OF ENZYME CATALYSIS

酶催化机制

• 批准号: 2468889
• 负责人: DEBRA DUNAWAY-MARIANO
• 金额: $ 28.02万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-02-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2468889
• 关键词: Pseudomonas; X ray crystallography; acid thiol ligase; active sites; biochemical evolution; bioenergetics; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate complex; hydro lyase; hydrolase; isomerase; site directed mutagenesis

DESCRIPTION: The long range goal of this project is to understand the process of evolution of new enzymatic activities. The system which will be used to study this process consists of the three enzymes of the 4-chlorobenzoate degrading pathway found in adapted soil bacteria. The proposed work is divided into three parts. These parts and their specific aims are as follows: Part I: Evolution of catalysis in 4-chlorobenzoyl-CoA dehalogenase. 1. Determine the energy profile for catalysis. 2. Measure the contributions of active site residues to the free energy of ground states and transition states in catalysis. 3. Test interchange of dehalogenase, crotonase and delta-3-cis, delta-2-trans enoyl-CoA isomerase activities within the dehalogenase scaffold. 4. Test in vivo evolution of 4-fluorobenzoyl-CoA activity and 2-chlorobenzoyl-CoA activity from the 4-chlorobenzoyl-CoA progenitor and in vitro evolution by random mutagenesis and by rational design. Part II: Mechanism and structure of 4-chlorobenzoate:CoA ligase and 4-hydroxybenzoyl-CoA thioesterase. 5. Complete the x-ray structural determination of the crystalline thioesterase. 6. Measure the time course for a single turnover on the thioesterase and test for the formation of a covalent enzyme intermediate. 7. Determine the active site residues essential to substrate binding and catalysis by site directed mutagenesis. Part III: Mechanism and structure of 4-chlorobenzoate:CoA ligase. 8. Crystallize and determine the x-ray structure of the ligase from Pseudomonas and/or Alcaligenes. 9. Locate the CoA binding site by site-directed mutagenesis of conserved residues. 10. Complete the determination of the kinetic mechanism of ligase catalysis.

描述：该项目的长期目标是了解 新酶活性的进化过程。 该系统将是 用于研究该过程的酶由三种酶组成 在适应的土壤细菌中发现 4-氯苯甲酸酯降解途径。 这 拟议的工作分为三个部分。 这些部分及其具体 目标如下： 第一部分：4-氯苯甲酰辅酶A催化作用的演变 脱卤酶。 1. 确定催化的能量分布。 2. 测量 活性位点残基对地面自由能的贡献 催化中的状态和过渡态。 3. 测试互换 脱卤酶、巴豆酸酶和 δ-3-顺式、δ-2-反式烯酰辅酶 A 异构酶 脱卤酶支架内的活性。 4. 测试体内进化 4-氟苯甲酰基-CoA 活性和 2-氯苯甲酰基-CoA 活性 4-氯苯甲酰基-CoA祖细胞和随机诱变的体外进化 并通过合理的设计。 第二部分：机制和结构 4-氯苯甲酸酯：CoA 连接酶和 4-羟基苯甲酰基-CoA 硫酯酶。 5. 完成结晶硫酯酶的 X 射线结构测定。 6. 测量硫酯酶单次周转的时间过程并 测试共价酶中间体的形成。 7. 确定 对底物结合和位点催化至关重要的活性位点残基 定向诱变。 第三部分：机制和结构 4-氯苯甲酸酯：CoA 连接酶。 8. 结晶并测定X射线 来自假单胞菌属和/或产碱菌属的连接酶的结构。 9. 找到 通过保守残基的定点诱变形成 CoA 结合位点。 10. 完成连接酶催化动力学机制的测定。