Dynamic and stuctural investigations of the Fructose-2,6-Bisphosphatase - a NMR study using standard and novel methods.

The aim of the proposed project is to investigate the dynamic and structural properties of an enzyme by standard nuclear magnetic resonance (NMR) techniques, as well as novel methods partly already developed, partly to be developed. The protein to be studied is the fructose-2,6-bisphosphatase. This enzyme is important for the regulation of carbohydrate metabolic pathways, i.e. the reciprocal regulation of the two opposite carbohydrate metabolic pathways, glycolysis and glyconeogenesis. These two metabolic pathways mediated by hepatic 6-phosphofructo-2- kinase/fructose-2,6-bisphosphate (6-PF-2-K/Fru-2,6-P2 ) are the major mechanism for the control in energy storage and blood glucose levels in mammalian systems. Therefore the enzyme has been postulated to provide an important switching mechanism between the glycolytic and gluconeogenic pathways in liver. The cellular concentration of Fru-2,6-P2 is regulated by glucagon and insulin through phosphorylation and dephosphorylation of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P2 ase). A better understanding of its function might well have important therapeutic conse-quences for diabetes. After a complete sequential assignment, which is a prerequisite for further NMR studies we will address the following aspects of fructose-2,6-bisphosphatase: protein-protein interaction; proton exchange rates; protein bound water molecules; and solution structure. These investigations will partly be done by means of selective NMR experiments, as well as HEHAHA transfers. The proposed NMR methods are especially aimed at enhancing the sensitivity of coherence transfer in fast exchanging systems and simplifying spectra by a new way of selective excitation. The feasibility of the proposed project has already been demonstrated by preliminary experiments and simulations of the presented NMR experiments.

The bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBP-2), provides a switch between the glycolytic and gluconeogenic pathways in liver. Reciprocal regulation of these opposing metabolic pathways by hepatic PFK-2/FBP-2 is accomplished by way of fructose-2,6-bisphosphate (F-2,6-P2 ), a biofactor which has differential allosteric effects on the activities of 6-phosphofructo-1-kinase (PFK-1) and fructose-1,6- bisphosphatase (FBP-1). Under conditions of high F-2,6-P2 , the resulting activation of PFK-1 promotes glycolysis while the inhibition of FBP-1 diminishes gluconeogenesis. In the mammalian liver the F-2,6-P2 content is primarily determined by the serum levels of glucagon, insulin, and glucose via intra-cellular signalling pathways and the subsequent phosphorylation or dephosphorylation of PFK-2/FBP-2. The regulatory power of this system has been exploited to achieve significant improvement of glycemic control in rodent models of Types 1 and 2 diabetes mellitus. In all the NMR spectra acquired during the course of this work we were able to assign 93 % of the 250 amino acids which build up the FBP-2 domain. The success of targeting hepatic F-2,6-P2 content as a means to treat DM underscores the relevance of the sequential resonance assignments of the rat liver FBP-2 domain (residues 250-470 of the bifunctional enzyme) that we have focussed on. These data provide a crucial step in the continued use of NMR spectroscopy to understand the function of hepatic FBP-2 with an eye toward the design of pharmacological inhibitors that may result in new treatments for DM in humans.