Mechanisms of Allosteric Influence on Enzymes Activity

描述（由申请人提供）：对代谢控制以及对药物设计越来越重要的调节基序是酶活性的变构修饰。这项研究计划的长期目标是了解变构调节的分子基础。目前，我们专注于系统中，变构配体实现其影响，通过改变其底物的酶的亲和力。来自各种细菌来源的磷酸果糖激酶（PFK）将作为模型系统进行研究，并作为酶的真核形式的最终研究的前奏。这些酶是同源四聚体，每个亚基含有单个活性位点和单个变构位点。尽管这种相对简单的组成，10个独特的成对变构相互作用可能存在。已经产生了这些酶的杂交形式，其分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。我们提出解决四个问题的广泛相关的许多变构酶。第一个问题是，在前面的授权项中确定的不同的异向性能量相互作用是否来自四聚体内的准独立相互作用途径。我们将解决这个问题，通过映射的途径，通过使用点突变引入到杂交选择性和评估的独立性，个别途径上的能量扰动。第二个问题是熵变是否有助于变构配体的作用与酶动力学的变化有关。这个问题将通过创建一个单一的变构相互作用和一个单一的位于一个已知的位置相对于相互作用的网站的杂合体。该物质将作为时间分辨荧光实验的报告者，旨在揭示其局部的流动程度。通过构建一个杂交体库，每个杂交体在不同的位置都有一个寡核苷酸，应该可以全面评估整个单个亚基的结构动力学变化。这些研究将通过类似的甲基-TROSY NMR实验来补充，该实验测量亮氨酸、异亮氨酸和缬氨酸残基的侧链动态。第三个问题是，当变构抑制剂或底物与激活剂结合时，酶的限制性结构形式是否揭示了导致抑制剂结合与底物结合之间拮抗作用的结构冲突。当抑制剂与原核PFK结合时发生的四级转移证实了这种主要的构象变化，并且将结合诱变、X射线晶体学、Fvrster共振能量转移和NMR方法对其进行研究。最后，将通过系统地表征各种变构配体类似物来研究对建立变构反应的性质和幅度很重要的变构配体的结构特征。如果要最终设计出靶向变构位点的药物，有必要了解这些是否是配体结构的可分离特征。公共卫生相关性：变构酶越来越多地被选择作为基于结构的药物设计的靶点，然而对与变构行为的原因相关的许多基本问题的理解仍然缺乏。我们的目标是提高这种理解，从而提高这些药物设计工作的成功前景。

DESCRIPTION (provided by applicant): 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 allosteric site per subunit. Despite this relatively simple composition, 10 unique pair-wise allosteric interactions can potentially exist. Hybrid forms of these enzymes 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 tryptophane located in a known position relative to the interacting sites. The tryptophane 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 tryptophane 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, Fvrster 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. PUBLIC HEALTH RELEVANCE: Allosteric enzymes are increasingly being selected as targets for structure-based drug design, yet understanding of many fundamental issues related to the causes of allosteric behavior is still lacking. Our goal is to improve this understanding and thereby improve the prospects for success in these drug design efforts.