Structures of S. pombe phosphofructokinase in the F6P-bound and ATP-bound states.
Phosphofructokinase (Pfk1; EC 2.7.1.11) is the third enzyme of the glycolytic pathway catalyzing the formation of fructose-1,6-bisphosphate from fructose-6-phosphate (F6P) and ATP. Schizosaccharomyces pombe Pfk1 is a homo-octameric enzyme of 800 kDa molecular weight, distinct from its yeast counterparts which are mostly hetero-octameric enzymes composed of two different subunits. Having an "open" conformation and a tendency to aggregate into higher oligomeric structures, the S. pombe enzyme shows similarities to the mammalian muscle Pfk1. It has been proposed that due to the distinct N-terminal region of the S. pombe subunit, the oligomeric organization of subunits in this enzyme is different from other yeast phosphofructokinases. Electron microscopy studies were carried out to reveal the quaternary structure of the homo-octameric Pfk1 from S. pombe in the F6P-bound and in the ATP-bound state. Random conical tilt data sets have been collected from deep stain preparations of the enzyme in both states. The 0 degrees tilt images have been separated into different classes and a 3D reconstruction has been calculated for each class from the high tilt images. Our results confirm the presence of a variety of views of the particle, most of which can be interpreted as views of the molecule rotating around its long axis. Despite the biochemical differences, the structure of phosphofructokinase from S. pombe in the presence of either F6P or ATP is similar to the hetero-octameric structure of phosphofructokinase from Saccharomyces cerevisiae. The molecule can be described as composed of two subdomains, connected by two well-defined densities. We have been able to establish a correlation between the kinetic behavior and the structural conformation of Pfk1.
Benjamin
Shaun
S
University of Vermont, College of Medicine, Department of Molecular Physiology and Biophysics, Burlington, VT 05405, USA.
Radermacher
Michael
M
Bär
Jörg
J
Edelmann
Anke
A
Ruiz
Teresa
T
D000255:Adenosine Triphosphate / Q000737:chemistry; D005636:Fructosephosphates / Q000737:chemistry; D008854:Microscopy, Electron; D010732:Phosphofructokinase-1 / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry*
D005636:Fructosephosphates; D029702:Schizosaccharomyces pombe Proteins; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1