EUKARYOTIC PHOSPHOFRUCTOKINASE: STRUCTURE/FUNCTION

真核磷酸果糖激酶：结构/功能

• 批准号: 7478035
• 负责人: TERESA RUIZ
• 金额: $ 23.06万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7478035
• 关键词: Adenosine Monophosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Affinity; Bacteria; Behavior; Binding; Biochemical; Catalysis; Catalytic Domain; Cells; Clinical; Complex; Cryoelectron Microscopy; Depth; Disease; Dissociation; Electron Microscopy; Enzymes; Eukaryota; Eukaryotic Cell; Exhibits; Family; Fission Yeast; Functional disorder; Glycolysis; Hemolytic Anemia; Knowledge; Life; Modeling; Negative Staining; Non-Insulin-Dependent Diabetes Mellitus; Numbers; Organism; Pathway interactions; Play; Production; Reaction; Regulation; Research; Resolution; Respiration; Role; Saccharomyces cerevisiae; Structure; Structure-Activity Relationship; Syndrome; Techniques; analog; cancer cell; enzyme mechanism; enzyme structure; fructose 2,6-diphosphate; fructose-6-phosphate; image processing; inhibitor/antagonist; insight; malignant muscle neoplasm; novel; particle; research study; size; sugar

DESCRIPTION (provided by applicant): Glycolysis and respiration are the main pathways for energy production in living cells. Although respiration is mostly favored by multicellular organisms, muscle and cancer cells behave more like unicellular organisms using glycolysis as their main energy pathway. Deficiencies and dysfunction of glycolytic enzymes, in particular phosphofructokinase (Pfk-1), results in severe clinical syndromes and diseases (e.g., Hemolytic anemia, Tauri's disease, non-insulin dependent diabetes mellitus). Pfk-1 plays a key role in the regulation of the glycolytic pathway and its activity is controlled by a large number of allosteric effectors (~20 in eukaryotes vs 2 in bacteria). The reaction catalyzed by this enzyme represents the first irreversible step specific for glycolysis. During the past twenty years, large efforts have been devoted to comprehend the mechanisms of catalysis and regulation of phosphofructokinase. Even though the information about the bacterial enzyme represents a great advancement in understanding this step of the glycolytic pathway, our knowledge of this enzyme is still quite limited for higher organisms. Eukaryotic Pfk-1's not only differ in size and oligomerization state, but they also exhibit a concentration dependent association-dissociation behavior and a far more complex regulatory mechanism. Moreover, the structures of the eukaryotic enzymes are still unknown, in most cases due to the lack of good quality crystals for x-ray analysis. The aim of this research is to analyze the structure of Pfk-1 from eukaryotic organisms (S. cerevisiae, S. pombe) in the presence of different combinations of effectors and substrates by novel techniques of cryo-electron microscopy of single particles and image processing, and by fitting x-ray models to the electron microscopy structures. These studies will provide significant new information regarding the structure/function relationship of the mechanism of catalysis and regulation of phosphofructokinase in eukaryotic organisms.

描述（由申请人提供）：糖酵解和呼吸是活细胞能量产生的主要途径。尽管呼吸主要受多细胞生物的青睐，但肌肉和癌细胞的表现更像是使用糖酵解作为主要能量途径的单细胞生物。糖酵解酶的缺乏和功能障碍，特别是磷酸果果因酶（PFK-1），导致严重的临床综合征和疾病（例如溶血性贫血，陶里氏病，非胰岛素依赖蛋白糖尿病）。 PFK-1在调节糖酵解途径中起关键作用，其活性受大量的变构效应子的控制（在真核生物中约为细菌中的2个）。该酶催化的反应代表了糖酵解的第一个不可逆的步骤。在过去的二十年中，已经大力努力理解磷酸果糖酶的催化和调节机制。即使有关细菌酶的信息在理解糖酵解途径的这一步骤方面取得了巨大的进步，但我们对这种酶的了解对于更高的有机体仍非常有限。真核PFK-1的大小和低聚状态不同，而且还表现出浓度依赖于浓度的关联分解行为和更为复杂的调节机制。此外，由于缺乏用于X射线分析的高质量晶体，真核酶的结构仍然未知。这项研究的目的是通过在存在不同组合的效应子和底物组合的情况下，通过单个颗粒和图像处理的冷冻电子显微镜的新技术，以及通过将X射线模型拟合电子显微镜结构的新型技术来分析效应子和底物的不同组合中的PFK-1结构。这些研究将提供有关真核生物中催化机理和调节磷脂酶的结构/功能关系的重要新信息。