Organization and Structural Features of Phosphofructokinase and other Glycolytic Enzymes to Meet their Role in Energy Metabolism

Although glycolytic enzymes can be isolated as cytosolic components, some of them are known to bind to proteins from the cytoplasm and other cellular compartments, such as the mitochondria, the nucleus, or the plasma membrane. In some cases these protein-protein interactions are considered significant for the regulation of energy metabolism, as well as for the modulation of other cell functions. Among glycolytic enzymes, phosphofructokinase (PFK) is thought to play a fundamental role in the control of this pathway, because of the number of metabolic signals that can regulate its complex allosteric behavior. No X-ray structure of a characteristic eukaryotic PFK is available yet. However, sequence data indicated that the eukaryotic enzyme originated by duplication, fusion and divergence of an ancestral prokaryotic gene, such that the duplicated fructose 6-phosphate catalytic site in the C-terminal half became an allosteric site for the activator fructose 2,6-bisphosphate. It has been suggested that both sites are shared in the interface between subunits aligned in an antiparallel orientation. To test the contribution of each terminal domain to these two binding sites, chimeric mammalian PFKs involving exchange of their terminal domains have been found to exhibit affinity properties for fructose 6-phosphate and fructose 2,6-bisphosphate that resembled those of the native isozyme that donated the N-terminal half and the C-terminal half, respectively. Further mutational analyses of muscle PFK led us to investigate the composition of the fructose phosphate binding sites and to gain insight into the structural organization of eukaryotic PFK. Data will also be presented evidencing the relationship between mitochondrial function and the operation of glycolysis, as observed after impairment of mitochondrial DNA transcription.