STRUCTURE/FUNCTION ANALYSIS OF ENZYMES

酶的结构/功能分析

• 批准号: 6180678
• 负责人: Hazel M. Holden
• 金额: $ 20.05万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180678
• 关键词: X ray crystallography; active sites; bacterial proteins; carbamoylphosphate synthase; enzyme activity; enzyme structure; enzyme substrate; esterase; fatty acid synthase; oxygenases; protein structure function; site directed mutagenesis; thioester

DESCRIPTION: High resolution x-ray crystallographic studies, in combination with detailed kinetic analyses, have yielded a wealth of understanding with respect to enzyme structure and function relationships. The goal of this application is to understand, on a detailed molecular level, the catalytic mechanisms of four enzymatic systems: phosphotriesterase from Pseudomonas diminuta which hydrolyzes certain pesticides and nerve agents, 4-chlorobenzoyl CoA dehalogenase and 4-hydroxybenzoyl CoA thioesterase from Pseudomonas sp. strain CBS-3 which are involved in the pathway that converts 4-chlorobenzoate to 4-hydroxybenzoate, and carbamoyl phosphate synthetase which produces the high energy intermediate carbamoyl phosphate. All of the catalytic mechanisms for these enzymes are believed to proceed through nucleophilic attacks. These enzymes were chosen for investigation due to their interesting biochemical features. The phosphotriesterase utilizes a binuclear metal center for activity while the dehalogenase catalyzes nucleophilic aromatic substitutions. The reaction mechanism of the thioesterase most likely proceeds through a nucleophilic attack but the amino acid sequence of the protein suggests that it is not similar to other thioesterases studied thus far. In the case of carbamoyl phosphate synthetase, the reaction mechanism proceeds via three nucleophilic attacks leading to two reactive intermediates, carboxyphosphate and carbamate. For these proteins, a combination of site-directed mutagenesis experiments and x-ray crystallographic analyses will be employed in order to more fully characterize their three-dimensional architectures, active site geometries, catalytic mechanisms, and substrate specificities.

描述：结合高分辨率 X 射线晶体学研究 通过详细的动力学分析，获得了丰富的理解 关于酶的结构和功能关系。 此举的目标 应用是在详细的分子水平上了解催化 四种酶系统的机制：来自假单胞菌的磷酸三酯酶 diminuta 可以水解某些杀虫剂和神经毒剂， 4-氯苯甲酰辅酶A脱卤酶和4-羟基苯甲酰辅酶A硫酯酶 假单胞菌属。 菌株 CBS-3 参与以下途径 将 4-氯苯甲酸酯转化为 4-羟基苯甲酸酯和氨基甲酰磷酸 产生高能中间体氨基甲酰磷酸的合成酶。 据信这些酶的所有催化机制都在进行 通过亲核攻击。 选择这些酶进行研究 由于它们有趣的生化特征。 磷酸三酯酶 利用双核金属中心进行活性，而脱卤酶 催化亲核芳香取代。 反应机理 硫酯酶很可能通过亲核攻击进行，但 该蛋白质的氨基酸序列表明它与其他蛋白质并不相似 迄今为止研究过的硫酯酶。 在氨基甲酰磷酸的情况下 合成酶，反应机制通过三个亲核攻击进行 产生两种反应中间体：羧基磷酸盐和氨基甲酸盐。 为了 这些蛋白质，结合定点诱变实验和 将采用 X 射线晶体学分析，以便更充分地 描述其三维架构、活性位点几何形状、 催化机制和底物特异性。