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Mechanisms of Allosteric Influence on Enzymes Activity

变构对酶活性的影响机制

基本信息

批准号：
8197459
负责人：
GREGORY Duncan REINHART
金额：
$ 38.39万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-08-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8197459
关键词：
Active Sites Address Affinity Allosteric Regulation Allosteric Site Amino Acids Attention Bacteria Behavior Binding Biochemical Biological Models Characteristics Commit Complement Complex Conflict (Psychology)Coupling Drug Delivery Systems Drug Design Energy Transfer Entropy Enzymes Fluorescence Free Energy Glycolysis Goals Grant Hybrids Individual Isoleucine Leucine Libraries Ligand Binding Ligands Light Maps Measures Metabolic Control Modification Molecular Mutagenesis Nature Pathway interactions Physiological Play Point Mutation Positioning Attribute Property Protein Dynamics Regulation Relative (related person)Reporter Research Role Side Signal Transduction Site Source Structure Sum System Time Tryptophan Valine Work X-Ray Crystallography analog base design enzyme activity enzyme model experience improved inhibitor/antagonist interest programs protein structure research study response success

项目摘要

A regulatory motif of fundamental importance to metabolic control, and increasingly to drug design, is the allosteric modification of enzyme activity. The long-term goal of this research program is to understand the molecular basis for allosteric regulation. Currently we are focused on systems in which allosteric ligands achieve their effects by altering the affinity of the enzyme for its substrate. Phosphofructokinase (PFK) from a variety of bacterial sources will be investigated as model systems and as a prelude to eventual studies of eukaryotic forms of the enzyme. These enzymes are homotetramers containing a single active site and a single allsoteric site per subunit. Despite this relatively simple composition, 10 unique pair-wise allosteric interactions can potentially exist. Hybrid forms of these enzmes have been produced that isolate individual allosteric interactions, the sum of which quantitatively explain the allosteric response in the native tetramer. We propose to address four questions of broad relevance to many allosteric enzymes. The first question is whether the different heterotropic energetic interactions identified in the previous grant term arise from quasi- independent interaction pathways within the tetramer. We will address this question by mapping the pathways through the use of point mutations introduced selectively into the hybrids and assessing the independence of the energetic perturbation on individual pathways. The second question is whether the entropy change that contributes to the action of an allosteric ligand is related to changes in enzyme dynamics. This question will be approached by creating hybrids with a single allosteric interaction and a single tryptophan located in a known position relative to the interacting sites. The tryptophan will serve as a reporter for time-resolved fluorescence experiments designed to reveal its local degree of mobility. By constructing a library of hybrids, each with a tryptophan in a different position, a comprehensive assessment of structural dynamics changes throughout a single subunit should be possible. These studies will be complemented by analogous methyl-TROSY NMR experiments measuring the side-chain dynamics of leucine, isoleucine, and valine residues. The third question relates to whether limiting structural forms of an enzyme, obtained when either allosteric inhibitors or substrates and activators bind, reveal the structural conflict that causes the antagonism between the binding of inhibitor and the binding of substrate. The quaternary shift that occurs when an inhibitor binds to prokaryotic PFK exemplifies such a major conformational change, and it will be studied with a combination of mutagenesis, X-ray crystallography, F¿rster Resonance Energy Transfer, and NMR approaches. Finally, the structural features of the allosteric ligand that are important to establishing the nature and magnitude of the allosteric response will be investigated by systematically characterizing various allosteric ligand analogs. It is necessary to understand whether these are separable features of ligand structure if one is to eventually design drugs that target allosteric sites.

对代谢控制以及对药物设计日益重要的一个调节基序是酶活性的变构修饰。该研究计划的长期目标是了解变构调节的分子基础。目前我们专注于变构配体的系统通过改变酶与其底物的亲和力来实现其效果。磷酸果糖激酶 (PFK) 各种细菌来源将作为模型系统进行研究，并作为最终研究的前奏该酶的真核形式。这些酶是同源四聚体，含有单个活性位点和单个每个亚基的变位点。尽管组成相对简单，但 10 个独特的成对变构相互作用可能存在。这些酶的混合形式已经产生，可以隔离个体变构相互作用，其总和定量地解释了天然四聚体中的变构反应。我们建议解决与许多变构酶广泛相关的四个问题。第一个问题是先前资助期限中确定的不同异方能量相互作用是否来自准四聚体内独立的相互作用途径。我们将通过映射来解决这个问题通过使用选择性引入杂交体的点突变并评估个体路径上能量扰动的独立性。第二个问题是是否有助于变构配体作用的熵变化与酶动力学的变化有关。这个问题将通过创建具有单一变构相互作用和单一色氨酸位于相对于相互作用位点的已知位置。色氨酸将作为用于时间分辨荧光实验的报告器，旨在揭示其局部的迁移程度。经过构建杂交文库，每个文库在不同位置都有色氨酸，进行全面评估整个单个亚基的结构动力学变化应该是可能的。这些研究将辅以类似的甲基-TROSY NMR 实验测量亮氨酸的侧链动力学，异亮氨酸和缬氨酸残基。第三个问题涉及是否限制酶的结构形式，当变构抑制剂或底物和激活剂结合时获得，揭示了结构冲突引起抑制剂的结合和底物的结合之间的拮抗作用。第四纪位移当抑制剂与原核 PFK 结合时发生的现象就是这种主要构象变化的例证，并且它将结合诱变、X 射线晶体学、F¿rster 共振能量转移和核磁共振方法。最后，变构配体的结构特征对于建立变构反应的性质和幅度将通过系统地表征各种别构配体类似物。有必要了解这些是否是配体结构的可分离特征如果要最终设计出针对变构位点的药物。