Objectives are to understand the allosteric control of gluconeogenesis by the enzyme, fructose-1,6-bisphosphatase, in molecular detail. Specific aims include: (1) development of a system for the expression of recombinant pig kidney fructose-1,6- bisphosphatase at high levels without contamination from the fructose-1,6-bisphosphatase of the host system, (2) cloning and overexpression of the E. coli fructose-1,6-bisphosphatase for comparison studies with the pig kidney enzyme, (3) use of the crystal structures of pig kidney fructose-1,6-bisphosphatase in the T and R states, and functional studies employing single site mutants of the E. coli and pig kidney enzymes to begin to determine the molecular basis of the switch between the allosteric states, and (4) determination of how the regulatory effector AMP alters the activity of the enzyme. %%% The degradation and biosynthesis of glucose are perhaps the most important of all metabolic pathways because glucose is the primary energy source for the brain. Given that the body does not store more than a one day supply of glucose, survival depends upon the ability to synthesize glucose via gluconeogenesis, after the stored supply of glucose is depleted. These studies will be aimed at understanding the function of the enzyme fructose-1,6- bisphosphatase, an enzyme that exerts metabolic control over gluconeogenesis. The enzyme's structure will be selectively modified via site directed mutagenesis and biophysical techniques will be utilized to characterize the resulting changes in structure. These structure changes will then be correlated with alterations in biological function. In this way specific aspects of the structure of the enzyme will be correlated with particular functions.
