EUKARYOTIC PHOSPHOFRUCTOKINASE: STRUCTURE/FUNCTION

描述(申请人提供)：糖酵解和呼吸作用是活细胞产生能量的主要途径。虽然呼吸作用主要是多细胞生物所青睐的，但肌肉和癌细胞的行为更像是以糖酵解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，特别是磷酸果糖激酶(PFK-1)，会导致严重的临床症状和疾病(例如，溶血性贫血、Tauri病、非胰岛素依赖型糖尿病)。PFK-1在糖酵解途径的调节中起着关键作用，其活性受大量的变构效应调控(真核生物中约20个，细菌中约2个)。该酶催化的反应是糖酵解的第一个不可逆步骤。在过去的二十年里，人们致力于了解磷酸果糖激酶的催化和调节机制。尽管关于细菌酶的信息在理解糖酵解途径的这一步方面取得了很大的进步，但对于高等生物来说，我们对这一酶的了解仍然相当有限。真核细胞pFK-1‘S不仅在大小和寡聚状态上存在差异，而且表现出浓度依赖的缔合-解离行为和更为复杂的调控机制。此外，在大多数情况下，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。本研究的目的是通过单颗粒冷冻电子显微镜和图像处理的新技术，并通过将X射线模型与电子显微镜结构相匹配，来分析真核生物(酿酒酵母、庞氏葡萄球菌)在不同效应物和底物组合存在下的PFK-1的结构。这些研究将为真核生物中磷酸果糖激酶的催化和调控机制的结构/功能关系提供重要的新信息。

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.