Mechanisms of Allosteric Influence on Enzymes Activity

描述(由申请人提供)：酶活性的变构修饰对新陈代谢控制，以及越来越多的药物设计具有重要的调节作用。该研究计划的长期目标是了解变构调节的分子基础。目前，我们关注的是变构配体通过改变酶对底物的亲和力来实现其作用的体系。来自各种细菌来源的磷酸果糖激酶(PFK)将被作为模型系统进行研究，并作为最终研究该酶的真核形式的前奏。这些酶是同源异构体，每个亚基包含一个活性部位和一个变构部位。尽管组成相对简单，但可能存在10种独特的成对变构相互作用。这些酶的杂交形式已经被产生，它们分离出个体变构相互作用，它们的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，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四聚体中的准独立相互作用路径。我们将通过使用选择性地引入到杂交种中的点突变来绘制路径图，并评估单个路径上能量扰动的独立性，从而解决这个问题。第二个问题是，有助于变构配体作用的熵变化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置的已知位置的单一色氨酸的杂化来解决。色氨酸将作为时间分辨荧光实验的记者，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都有一个色氨酸，应该可以全面评估整个单个亚基的结构动力学变化。这些研究将得到类似的甲基-TROSY核磁共振实验的补充，这些实验测量亮氨酸、异亮氨酸和缬氨酸残基的侧链动力学。第三个问题是，当变构抑制剂或底物和激活剂结合时，酶的限制结构形式是否揭示了导致抑制剂结合和底物结合之间的拮抗作用的结构冲突。当抑制剂与原核生物PFK结合时发生的四元位移就是这种主要构象变化的例证，将结合突变、X射线结晶学、Fvrster共振能量转移和核磁共振方法进行研究。最后，将通过系统地表征各种变构配体类似物来研究变构配体的结构特征，这些结构特征对于确定变构反应的性质和大小很重要。如果要最终设计以变构为靶点的药物，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为基于结构的药物设计的靶标，但对与变构行为原因相关的许多基本问题的了解仍然不足。我们的目标是提高这种认识，从而提高在这些药物设计工作中取得成功的前景。

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.