EUKARYOTIC PHOSPHOFRUCTOKINASE: STRUCTURE/FUNCTION

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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.