STRUCTURE AND FUNCTION IN ENZYME CATALYSIS

酶催化的结构和功能

• 批准号: 3291829
• 负责人: JEREMY R KNOWLES
• 金额: $ 19.39万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3291829
• 关键词: X ray crystallography; acidity /alkalinity; biochemical evolution; chemical structure function; chickens; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; genetic manipulation; molecular genetics; muscle; nuclear magnetic resonance spectroscopy; operon; point mutation; protein engineering; site directed mutagenesis; triose phosphate isomerase; yeasts

Of all enzymes, triosephosphate isomerase is one of the most completely defined in both kinetic and structural terms. The complete free energy profile was established in our laboratory in 1976, and the crystal structures of isomerases from both chicken muscle and from yeast have been solved to high resolution. Structures of complexes of the enzyme with both substrate and inhibitors are also available. From this firm basis of structure:function correlation, we aim to use both site-directed mutagenesis and classical genetic selection to define the nature of the three catalytic elements (the basic residue Glu-165, the acidic residues His-95 and Lys-13, and the 'loop' that closes on bound substrate) and delineate their contribution to the catalytic act. We shall evaluate the complete energetics for each mutant that we generate, and we aim (in collaboration with the crystallographers, and by NMR, FTIR, etc.) to define the precise structures of these mutants. Finally, starting with mutants that we have already produced, the activity of which has been severely reduced, we shall select for more active second site suppressor mutations, in order to illuminate the evolutionary path forward and to test, for the first time, the theories of the development of the catalytic function of enzymes.

在所有酶中，三尿磷酸异构酶是最完全的酶之一 以动力学和结构术语定义。 完整的自由能 配置文件是在1976年在我们的实验室建立的，晶体 来自鸡肉和酵母异构酶的结构已经 解决高分辨率。 酶的复合物结构 还提供底物和抑制剂。 从这个坚定的基础 结构：功能相关性，我们的目标是使用两个站点定向 诱变和经典遗传选择来定义 三个催化元素（碱性残基GLU-165，酸性残基 His-95和Lys-13，以及在绑定的底物上关闭的“循环”）和 描述他们对催化法的贡献。 我们将评估 为我们产生的每个突变体的完整能量学，我们的目标（在 与Crystallographter的合作，以及NMR，FTIR等）来定义 这些突变体的精确结构。 最后，从突变体开始 我们已经产生了的活动，其活动严重 减少，我们将选择更活跃的第二个位点抑制突变， 为了阐明进化路径的前进并测试 第一次，催化功能的发展理论 酶。