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 crucial role in the regulation of glucose levels. It is the sole enzyme responsible for the synthesis and degradation of fructose-2,6-bisphosphate, a potent intracellular modulator of heptic carbon flux. Fructose- 2,6-biphosphate is an allosteric activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase. The metabolic effects of glucagons, via cAMP-dependent protein kinase (where cAMP is cyclic adenosine monophosphate), on hepatic carbon flux are mediated by the intracellular concentration of fructose-1,6-biphosphate. In diabetes, the excessive production of glucose by the liver is a major contributor to hyperglycemia, which leads to the major problems associated with the disease. The central role of fructose-2,6- biphosphate in control of hepatic glucose levels will be beneficial to the diabetic patient. This group is investigating the efficacy of targeting the biphosphatase domain of the bifunctional enzyme for inhibition, and thereby increasing the fructose-2,6-biphosphate levels. The current model for an inhibited biphosphate has the phospho-accepting histidine mutated to alanine. Overexpression of this mutant in murine models of diabetes using an adenovirus vector has resulted in a normalization of blood glucose levels.

The Lange group is using Supercomputing Institute resources to process and display nuclear magnetic resonance data on the biphosphatase domain of bifunctional enzyme, in order to characterize the active site and defining the catalytic mechanism(s). The group has identified the roles of the catalytic histidines and an active site glutamatic acid residue. Using 13_C and 15_N labeling of the histidine ring together with mutagenesis, the protonation and tautomeric states of the histidines and perturbations of the active site cause by mutating the glutamatic acid to alanine were determined.

The group’s present work is pursuing the sequential assignment of the biphosphate domain that will facilitate solving a solution structure and further biophysical characterizations. Concurrently, a spectroscopic investigation of the arginine residues in the active site utilizing specific labeling of the histidines and arginines is proceeding. Knowledge of this important component of gluconeogenic/glycolytic flux helps provides the basis for the rational design of specific inhibitors of the hisphosphatase activity of heptic 6-phosphofructo- 2-kinase/fructose-2,6-biphosphate.

Research Group and Collaborator

Matthew H. Devany, Graduate Student Researcher
Dave Okar, Research Associate
Klaus Zangger, Visiting Researcher