EUKARYOTIC PHOSPHOFRUCTOKINASE: STRUCTURE/FUNCTION

描述（由申请人提供）：糖酵解和呼吸是活细胞中能量产生的主要途径。虽然多细胞生物更喜欢呼吸作用，但肌肉细胞和癌细胞的行为更像单细胞生物，它们以糖酵解为主要的能量途径。糖酵解酶，特别是磷酸果糖激酶（Pfk-1）的缺乏和功能障碍导致严重的临床综合征和疾病（例如，溶血性贫血、金牛座病、非胰岛素依赖型糖尿病）。Pfk-1在糖酵解途径的调控中起关键作用，其活性受大量变构效应物控制（真核生物约20个，细菌约2个）。这种酶催化的反应是糖酵解的第一个不可逆步骤。在过去的二十年中，人们对磷酸果糖激酶的催化和调控机制进行了大量的研究。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，但我们对这种酶的了解对于高等生物来说仍然相当有限。真核生物的Pfk-1不仅在大小和寡聚化状态上存在差异，而且还表现出浓度依赖的结合-解离行为和更为复杂的调控机制。此外，真核酶的结构仍然是未知的，在大多数情况下，由于缺乏高质量的晶体用于x射线分析。本研究的目的是分析真核生物（S. cerevisiae， S. pombe）在不同效应器和底物组合下Pfk-1的结构，采用单粒子冷冻电镜和图像处理的新技术，并将x射线模型拟合到电子显微镜结构中。这些研究将为真核生物磷酸果糖激酶催化调控机制的结构/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.