MECHANISM OF ALLOSTERIC INFLUENCE ON ENZYME ACTIVITY

变构对酶活性的影响机制

• 批准号: 3282622
• 负责人: GREGORY Duncan REINHART
• 金额: $ 14万
• 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/3282622
• 关键词: 6 phosphofructokinase; Bacillus stearothermophilus; Escherichia coli; acidity /alkalinity; allosteric site; carbamoylphosphate synthase; chemical aggregate; conformation; dogs; 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.

对新陈代谢控制至关重要的一个调控主题是 酶活性的变构修饰。长期目标 这一应用的目的是增加我们对机理的理解 变构配体通过什么修饰酶的活性 通过与从活性部位移除的酶上的部位结合。在……里面 我们特别感兴趣的是变构配体 通过改变酶对其亲和力来实现其效果 底物。 我们特别建议研究三种不同的变构系统： 原核生物磷酸果糖激酶(PFK)的分离和鉴定 嗜热脂肪芽孢杆菌；氨基甲酰磷酸合成酶 (CPS)和依赖NAD的异柠檬酸脱氢酶(ICDH) 从牛肉心脏线粒体中提取。所有这些酶都能满足 新陈代谢中重要的调节生态位。更重要的是， 然而，此应用程序的目标提供了机会 回答与许多人普遍相关的机械性问题 其他变构酶。通过研究这些酶，我们希望能够 回答有关变构反应的几个问题，包括 以下是：1)从以下角度来看待变构行为是否合适 已知两种X射线晶体结构的双态模型？2) 温度能改变变构的性质和大小吗？ 反应？3)两个结构相似的配体怎么会有相反的 当结合到相同的位置时，变构效应？以及4)是否可以单独 推导出定量模型来解释变构的作用 通过改变两个三级配基来达到其效果的配基 酶的构象和聚集状态？ 我们的方法从系统和彻底的链接函数开始 对变构配体作用的表征。这一分析 产生的术语不仅量化了底物的亲和力， 效应配体，还有变构的性质和大小 影响力。通过监视在这些参数中引起的变化 对实验变量的修改；例如配体结构， 酶结构、温度、酶浓度等；洞察力可以 得到了实验变量和实验变量之间的关系 变构配体的作用。 这种方法将得到物理研究的补充；值得注意的是 荧光强度、光谱分布、偏振和 本征色氨酸残基和共价态的寿命测量 附加的非本征荧光探针；以进一步定义结构 变构配体结合引起的后果。