MECHANISM OF ALLOSTERIC INFLUENCE ON ENZYME ACTIVITY

变构对酶活性的影响机制

• 批准号: 3282625
• 负责人: GREGORY Duncan REINHART
• 金额: $ 18.38万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-08-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282625
• 关键词: 6 phosphofructokinase; Bacillus stearothermophilus; Escherichia coli; acidity /alkalinity; allosteric site; carbamoylphosphate synthase; chemical aggregate; conformation; dogs; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate complex; fluorescence polarization; fluorescence spectrometry; fluorescent dye /probe; heart; isocitrate dehydrogenase; ligands; molecular rearrangement; protein structure function; radiotracer; temperature

A regulatory motif of fundamental importance to metabolic control is the allosteric modification of enzymatic activity. The long term objective of this application is to increase our understanding of the mechanisms by which allosteric ligands are able to modify enzymatic activity through binding to sites on the enzyme removed from the active site. In particular we are interested in systems in which the allosteric ligands achieve their effects by altering the affinity of enzyme for its substrate. We specifically propose to study three different allosteric systems: prokaryotic phosphofructokinase (PFK) isolated from both Escherichia coli and Bacillus stearothermophilus; carbamoyl phosphate synthetase (CPS) from E. Coli; and NAD-dependent isocitrate dehydrogenase (ICDH) obtained from beef heart mitochondria. All of these enzymes fulfill important regulatory niches in metabolism. Even more importantly for the objectives of this application, however, they present opportunities for answering mechanistic questions that have general relevance to many other allosteric enzymes. By studying these enzymes we hope to be able to answer several questions regarding allosteric response including the following: 1) Is it appropriate to view allosteric behavior in terms of a two-state model even when two X-ray crystal structures are known? 2) Can temperature alter the nature as well as the magnitude of allosteric response? 3) How can two structurally similar ligands have opposite allosteric effects when binding to the same site? and 4) Can a single quantitative model be derived to explain the actions of an allosteric ligand that achieves its effects by altering both the tertiary conformation and the aggregation state of an enzyme? Our approach begins with a systematic and thorough linked-function characterization of the actions of an allosteric ligand. This analysis yields terms that quantify not only the affinity of the substrate and effector ligand, but also the nature and magnitude of the allosteric influence. By monitoring the changes induced in these parameters by modifications of an experimental variable; such as ligand structure, enzyme structure, temperature, enzyme concentration, etc.; insight can be gained into the relationship between the experimental variable and the actions of the allosteric ligand. This approach will be complemented by physical studies; notably fluorescence intensity, spectral distribution, polarization, and lifetime measurements of intrinsic tryptophan residues and covalently attached extrinsic fluorescent probes; to further define the structural consequences resulting from allosteric ligand binding.

对代谢控制至关重要的调节基序是 酶活性的变构修饰。 长期目标 是为了增加我们对 变构配体能够通过其改变酶活性 通过与酶上从活性位点移除的位点结合。 在 特别是我们感兴趣的系统，其中变构配体 通过改变酶对其的亲和力来实现它们的作用。 衬底 我们特别建议研究三种不同的变构系统： 原核磷酸果糖激酶（PFK）分离自两种大肠杆菌 大肠杆菌和嗜热脂肪芽孢杆菌;氨甲酰磷酸合成酶 (CPS)来自大肠和NAD依赖性异柠檬酸脱氢酶（ICDH） 从牛心线粒体中提取的。 所有这些酶都能 代谢中重要的调节小生境。 更重要的是， 然而，该应用程序的目标提供了机会， 回答机械性的问题， 其他变构酶。 通过研究这些酶，我们希望能够 回答关于变构反应的几个问题，包括 以下：1）从以下方面看待变构行为是否合适： 即使已知两种X射线晶体结构，也能建立两态模型吗？ （二） 温度能改变变构的性质和大小吗 回应？ 3)两个结构相似的配体怎么会有相反的 当结合到同一位点时的变构效应？ （4）一个单一的 定量模型可以用来解释变构作用 配体，通过改变其三级结构 构象和聚集状态的一种酶？ 我们的方法从系统和彻底的链接功能开始 表征变构配体的作用。 该分析 产生的术语不仅量化了底物的亲和力， 效应配体，而且也变构的性质和幅度 影响力的社会 通过监测这些参数的变化， 实验变量的修饰;例如配体结构， 酶结构、温度、酶浓度等;洞察可以 得到实验变量和 变构配体的作用。 这一方法将得到物理研究的补充;特别是 荧光强度，光谱分布，偏振，和 固有色氨酸残基和共价色氨酸残基的寿命测量 连接外源性荧光探针;以进一步确定结构 由变构配体结合引起的后果。