Mechanisms of Allosteric Influence on Enzymes Activity

对于新陈代谢控制以及越来越多的药物设计来说，一个基本重要的调控主题是 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。目前我们关注的是变构配体的体系 通过改变酶对其底物的亲和力来实现它们的效果。磷酸果糖激酶(PFK) 各种细菌源将作为模型系统进行调查，并作为最终研究的前奏。 真核形式的酶。这些酶是同源异构体，只包含一个活性部位和一个 每个亚基的异温作用部位。尽管这种相对简单的组成，10个独特的成对变构 互动可能存在。已经产生了这些酶的杂交形式，以分离个体 变构相互作用，它们的总和定量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 在前一个授权期中确定的不同的各向异性能量相互作用是否源于准 四聚体内的独立相互作用途径。我们将通过将 通过使用有选择地引入杂交种的点突变和评估 独立于个体路径上的能量扰动。第二个问题是 有助于变构配体作用的熵变与酶动力学的变化有关。 这个问题将通过创造具有单一变构相互作用和单一变构相互作用的杂化来解决 色氨酸位于相对相互作用部位的已知位置。色氨酸将作为一种 记者进行了时间分辨荧光实验，旨在揭示其局部的流动性程度。通过 构建一个杂交体文库，每个杂交体在不同的位置有一个色氨酸，一个全面的评估 整个单个亚单位的结构动力学变化应该是可能的。这些研究将是 辅以测量亮氨酸侧链动力学的类似的甲基-TROSY核磁共振实验， 异亮氨酸和缬氨酸残基。第三个问题涉及酶的限制性结构形式， 当变构抑制剂或底物与激活剂结合时获得的，揭示了 导致抑制剂结合和底物结合之间的拮抗作用。第四纪的移位 当一种抑制剂与原核生物的PFK结合时就会发生这种主要的构象变化，它将 结合诱变、X射线结晶学、F？rster共振能量转移和 核磁共振方法。最后，研究了变构配体的结构特征，这些结构特征对建立构效关系具有重要意义。 变构反应的性质和大小将通过系统地表征各种 变构配体类似物。有必要了解这些是否是配体结构的可分离特征 如果一个人最终要设计出针对变构部位的药物。

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.