title: The 10.8-A structure of Saccharomyces cerevisiae phosphofructokinase determined by cryoelectron microscopy: localization of the putative fructose 6-phosphate binding sites.
abstract: Phosphofructokinase plays a key role in the regulation of the glycolytic pathway and is responsible for the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Although the structure of the bacterial enzyme is well understood, the knowledge is still quite limited for higher organisms given the larger size and complexity of the eukaryotic enzymes. We have studied phosphofructokinase from Saccharomyces cerevisiae in the presence of fructose 6-phosphate by cryoelectron microscopy and image analysis of single particles and obtained the structure at 10.8A resolution. This was achieved by optimizing the illumination conditions to obtain routinely 8-A data from hydrated samples in an electron microscope equipped with an LaB(6) and by improving the image alignment techniques. The analysis of the structure has evidenced that the homology of the subunits at the sequence level has transcended to the structural level. By fitting the X-ray structure of the bacterial tetramer into each dimer of the yeast octamer the putative binding sites for fructose 6-phosphate were revealed. The data presented here in combination with molecular replacement techniques have served to provide the initial phases to solve the X-ray structure of the yeast phosphofructokinase.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, VT 05405, USA. truiz@physiology.med.uvm.edu
authors.forename: Teresa
authors.initials: T
authors.lastname: Ruiz
authors.forename: Ingrid
authors.initials: I
authors.lastname: Mechin
authors.forename: Jörg
authors.initials: J
authors.lastname: Bär
authors.forename: Wojciech
authors.initials: W
authors.lastname: Rypniewski
authors.forename: Gerhard
authors.initials: G
authors.lastname: Kopperschläger
authors.forename: Michael
authors.initials: M
authors.lastname: Radermacher
mesh_terms: D001665:Binding Sites; D020285:Cryoelectron Microscopy; D004867:Equipment Design; D005636:Fructosephosphates / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification; D040681:Structural Homology, Protein
chemical_list: D005636:Fructosephosphates; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C027618:fructose-6-phosphate; D010732:Phosphofructokinase-1