Mechanisms of Allosteric Influence on Enzymes Activity

描述（由申请人提供）：酶活性的变构修饰是对代谢控制和药物设计具有重要基础意义的调控基序。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，我们关注的是通过改变酶对底物的亲和力来实现变构配体作用的系统。来自多种细菌来源的磷酸果糖激酶（PFK）将作为模型系统进行研究，并作为最终研究真核形式的酶的前奏。这些酶是同型四聚体，每个亚基含有一个活性位点和一个变构位点。尽管这种相对简单的组成，10种独特的成对变构相互作用可能存在。这些酶的杂交形式已经产生，分离出个体的变构相互作用，其总和定量地解释了天然四聚体的变构反应。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，在前一个授权期限中确定的不同的异向能相互作用是否来自四聚体内的准独立相互作用途径。我们将通过使用选择性引入杂交体的点突变来绘制路径，并评估能量扰动对单个路径的独立性来解决这个问题。第二个问题是导致变构配体作用的熵变是否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位于相对于相互作用位点的已知位置的单一色氨酸的杂交种来解决。色氨酸将作为时间分辨荧光实验的报告者，旨在揭示其局部迁移程度。通过构建一个杂化库，每个杂化库中色氨酸位于不同的位置，就可以对单个亚基的结构动力学变化进行全面评估。这些研究将辅以类似的甲基- trosy核磁共振实验，测量亮氨酸、异亮氨酸和缬氨酸残基的侧链动力学。第三个问题涉及到当变构抑制剂或底物与激活剂结合时获得的酶的限制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的结构冲突。当抑制剂与原核PFK结合时发生的四元转移就是这种主要构象变化的例证，我们将结合诱变、x射线晶体学、Fvrster共振能量转移和核磁共振方法对其进行研究。最后，通过系统地表征各种变构配体类似物，研究变构配体的结构特征，这些特征对确定变构反应的性质和幅度很重要。如果要最终设计针对变构位点的药物，有必要了解这些是否为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为基于结构的药物设计的靶标，然而对与变构行为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.