Alex J. Lange, Principal Investigator

Description of Bisphosphatase Activity Modulations of the C-terminus in the
Bifunctional Enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase

  The bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, plays a central role in the regulation of hepatic carbohydrate metabolism because it is the only enzyme that can synthesize or degrade fructose-2,6-bisphosphate. Fructose-2,6-bisphosphate is a potent allosteric activator of the glycolytic enzyme 6-phosphofructo-1-kinase and inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase. These allosteric effects of fructose-2,6-bisphosphate provide a switch between glycolysis (high fructose-2,6-bisphosphate) and gluconeogenesis (low fructose-2,6-bisphosphate) in the liver. The hepatic content of fructose-2,6-bisphosphate is determined by the relative kinase and bisphosphatase activities (K/B ratio) of the bifunctional enzyme which proceed at distinct sites. The bifunctional enzyme is as a homodimer and the K/B ratio is controlled by phosphorylation/dephosphorylation at Serine-32. When Serine-32 is phosphorylated by cAMP-dependent protein kinase the bisphosphatase is activated and the kinase inhibited producing a low K/B ratio and lowering the intracellular amount of fructose-2,6-bisphosphate. Dephosphorylation of Serine-32 by a protein phosphatase (PP2A) has the opposite effect. When the level of fructose-2,6-bisphosphate was modulated by adenovirus-mediated overexpression of the wild type or mutant bifunctional enzyme in cultured cells (FAO) or in the livers of living mice, the results confirmed that elevated fructose-2,6-bisphosphate levels could activate glycolysis and reduce hepatic glucose production. Because inappropriate production of glucose by the liver is a major contributor to the hyperglycemia that is characteristic of diabetes, these results strongly suggested that the liver bifunctional enzyme, especially the bisphosphatase active site, is a good target for the design novel antidiabetic agents.

  In their research, this group attempted to determine the solution structure of the separate bisphosphatase domain by multi-dimensional nuclear magnetic resonance (NMR) spectroscopy. The NMR results provided a necessary complement to the X-ray crystallographic structures and allowed us to determine the molecular mechanism of the bisphosphatase. A detailed description of how the bisphosphatase activity is modulated by the C-terminus of the enzyme: this is essential for a complete understanding of the bisphosphatase. The insight gained from the expanded NMR studies increases the understanding of how hepatic carbohydrate metabolism is controlled, facilitates the design of pharmacological inhibitors of the bisphosphatase, and aids in the reengineering of the bifunctional enzyme required for a gene therapy approach to ameliorate the hyperglycemia associated with diabetes.