STRUCTURE AND FUNCTION IN ENZYME CATALYSIS

酶催化的结构和功能

基本信息

批准号：
3291827
负责人：
JEREMY R KNOWLES
金额：
$ 18.4万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
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/3291827
关键词：
X ray crystallography acidity /alkalinity biochemical evolution chemical structure function enzyme structure enzyme substrate enzyme substrate analog genetic manipulation molecular genetics muscle nuclear magnetic resonance spectroscopy operon point mutation triose phosphate isomerase yeasts

项目摘要

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，磷酸三糖异构酶是最完全的酶之一从动力学和结构两个方面进行定义。完全自由能 1976年我们实验室建立了剖面，晶体来自鸡肌肉和酵母的异构酶的结构已被确定解决了高分辨率。酶与两者的复合物的结构底物和抑制剂也可用。从此坚实的基础结构：功能相关，我们的目标是同时使用定点诱变和经典遗传选择来定义三种催化元素（碱性残基 Glu-165、酸性残基 His-95 和 Lys-13，以及在结合底物上闭合的“环”）和描述他们对催化行为的贡献。我们将评估我们生成的每个突变体都有完整的能量学，我们的目标是（在与晶体学家合作，并通过 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生产了，其活动已受到严重影响减少，我们将选择更活跃的第二位点抑制突变，为了阐明前进的进化道路并进行测试首次提出了催化功能发展的理论酶。