Forum 08/17/2006

“The structure of S. cerevisiae phosphofructokinase in two functional states”

Teresa Ruiz, Assistant Professor – Department of Molecular Physiology and Biophysics, The University of Vermont

Phosphofructokinase (Pfk-1) plays a key role in the regulation of the glycolytic pathway. It catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-biphosphate in the presence of Mg-ATP. The combination of X-ray crystallographic and biochemical data has provided an understanding of the different conformational changes that occur between the active and inactive states of the bacterial enzyme and of the role played by its two effectors. Eukaryotic phosphofructokinases, however, are larger, more complex structures that due to the larger number of effectors that regulate them (over 20), exhibit a far more sophisticated regulatory mechanism.

Pfk-1 from S. cerevisiae is the best characterized among all the eukaryotic enzymes. This enzyme is a heterooctamer of 835 kDa, composed of four α and four β subunits. The 3D of this enzyme in the presence of F6P has been obtained by cryo-electron microscopy. Our results evidenced that nature used the bacterial tetramer as the structural building block for the higher eukaryotic enzymes and allowed the localization of the putative F6P catalytic binding sites. More over, this electron microscopy structure, in combination with molecular replacement has provided the initial phases to solve the x-ray structure of the 12S yeast truncated tetramer from in the presence of F6P.

Our goal is to establish a working model for the catalytic process of Pfk-1 in eukaryotes. We have calculated a 3D reconstruction from frozen-hydrated preparations of the enzyme in the presence of Mg-ATP and compared it with our 3D reconstruction of the enzyme in the presence of F6P. We have fitted the X-ray model of the two truncated tetramers in the presence of F6P into the calculated electron microscopy volume in the F6P-state and in the ATP-state to analyze the structural changes in the subunit at the atomic level.