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：高分辨率X射线晶体学研究，结合 详细的动力学分析，已经产生了丰富的理解， 酶的结构和功能关系。 这个目标 应用是为了在详细的分子水平上了解催化剂， 四种酶系统的作用机制：假单胞菌磷酸三酯酶 水解某些杀虫剂和神经毒剂的diminuta， 4-氯苯甲酰CoA脱卤酶和4-羟基苯甲酰CoA硫酯酶 假单胞菌属菌株CBS-3，其参与以下途径： 将4-氯苯甲酸酯转化为4-羟基苯甲酸酯， 合成酶，其产生高能量中间体氨甲酰磷酸。 这些酶的所有催化机制都被认为是 通过亲核攻击。 选择这些酶进行研究 因为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 催化亲核芳族取代。 的反应机理 硫酯酶最有可能通过亲核攻击进行， 蛋白质的氨基酸序列表明它与其他蛋白质不相似。 目前研究的硫酯酶。 在氨基甲酰磷酸盐的情况下 合成酶，反应机理通过三个亲核攻击进行 产生两种反应性中间体，羧基磷酸酯和氨基甲酸酯。 为 这些蛋白质，结合定点诱变实验， 将采用X射线晶体学分析，以便更全面地 表征它们的三维结构，活性部位的几何形状， 催化机制和底物特异性。