MECHANISM OF ALLOSTERIC INFLUENCE ON ENZYME ACTIVITY

变构对酶活性的影响机制

• 批准号: 3282618
• 负责人: GREGORY Duncan REINHART
• 金额: $ 10.16万
• 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 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282618
• 关键词: 6 phosphofructokinase; acidity /alkalinity; allosteric site; anions; carbohydrate metabolism; chemical association; enzyme substrate complex; fluorescent dye /probe; heart; isozymes; ligands; liver; molecular rearrangement; muscle; radiotracer

The long term objective of this application is to understand the molecular mechanisms by which allosteric ligands modify enzymatic behavior. Specifically, the allosteric properties of porcine heart fumarase, E. coli carbamoyl phosphate synthetase, bovine heart NAD-dependent isocitrate dehydrogenase, and rat liver phosphofructokinase will be investigated. Fumarase and carbamoyl phosphate synthetase are each regulated by allosteric modifiers that influence maximal velocity and/or Michaelis constants by altering the tertiary structure of the protein. The kinetic behavior of these enzymes will be studied using a thermodynamic linked-function approach. This approach provides for the quantitation of the dissociation constant of modifying ligand from free enzyme, the fractional influence that the modifying ligand has on maximal velocity, and the free energy of interaction between the substrate and allosteric ligand. These latter two parameters describe the efficacy of the allosteric ligand once bound. By observing the changes in these parameters produced by systematically altering the structure of the allosteric ligand or protein, we will be able to infer structural features of the ligand and protein important for these two separate facets of allosteric ligand action. This approach will be extended to investigate the mechanism of action of allosteric ligands that alter an enzyme's aggregation state as well and will be applied to a study of the regulation of isocitrate dehydrogenase by ADP, and phosphofructokinase by a wide variety of regulatory ligands. Isocitrate dehydrogenase should provide a straightforward example of the relationship between tertiary and quaternary levels of allosteric action, whereas phosphofructokinase represents a complex example of several different interdependent regulatory mechanisms. Despite this complexity, significant new insight should be obtained by quantitatively evaluating the pertinent free energy coupling parameters. This insight has been obscured in the past by the common assumption that the enzyme can exist in one of only two possible conformational states. This new insight is especially important in view of the vital role phosphofructokinase plays in carbohydrate homeostasis. The experimental methodologies required for these studies consist primarily of enzyme kinetics and fluorescence polarization measurements, the latter being performed on enzymes covalently labeled with a suitable extrinsic fluorescent probe.

该应用的长期目标是了解分子 变构配体改变酶行为的机制。 具体来说，猪心延胡索酸酶、大肠杆菌的变构特性 氨基甲酰磷酸合成酶，牛心 NAD 依赖性异柠檬酸 将研究脱氢酶和大鼠肝脏磷酸果糖激酶。 延胡索酶和氨基甲酰磷酸合成酶各自受以下调节 影响最大速度和/或米氏力的变构调节剂 通过改变蛋白质的三级结构来改变常数。 动力学 将使用热力学研究这些酶的行为 链接函数方法。 该方法提供了定量 修饰配体与游离酶的解离常数， 修饰配体对最大速度的影响分数，以及 底物和变构之间相互作用的自由能 配体。 后两个参数描述了效果 变构配体一旦结合。 通过观察这些参数的变化 通过系统地改变变构配体的结构而产生 或蛋白质，我们将能够推断配体的结构特征和 蛋白质对于变构配体作用的这两个不同方面很重要。 该方法将扩展到研究作用机制 变构配体也可以改变酶的聚集状态， 将应用于异柠檬酸脱氢酶调节的研究 ADP 和磷酸果糖激酶受到多种配体的调节。 异柠檬酸脱氢酶应该提供一个简单的例子 三级和四级变构作用之间的关系， 而磷酸果糖激酶代表了几种的复杂例子 不同的相互依存的调节机制。 尽管存在这种复杂性， 应该通过定量评估来获得重要的新见解 相关自由能耦合参数。 这种洞察力已被掩盖 过去普遍认为酶可以存在于其中之一 只有两种可能的构象状态。 这一新见解尤其 鉴于磷酸果糖激酶在 碳水化合物稳态。 这些研究所需的实验方法主要包括 酶动力学和荧光偏振测量，后者 对用合适的外源性共价标记的酶进行 荧光探针。