Proposed investigations focus on key enzymes of gluconeogenesis and glycolysis: fructose-1,6- bisphosphatase and hexokinase isoforms I and II. Fructose-1,6-bisphosphatase governs a control point in the biosynthetic pathway for glucose. The enzyme is subject to regulation by metabolites that bind to allosteric sites (AMP) or active sites (fructose 2,6-bisphosphate). The proposed model describes how small and localized conformational change, induced by the binding of allosteric or active-site inhibitors, triggers global conformational change in the tetrameric enzyme. Directed mutations, the formation of hybrid tetramers by subunit exchange, fluorescence spectroscopy, kinetics, and structure determinations by x-ray diffraction test the validity of proposed models for positive cooperativity in allosteric inhibition and for binding synergism between allosteric and active-site inhibitors. Research will define new sites from which an appropriate ligand can reinforce the action of physiological inhibitors. Inhibition of fructose-1,6-bisphosphate reduces levels of serum glucose in rats. Reductions in such levels in humans would ameliorate degenerative diseases associated with type 2 diabetes. The release of hexokinase isoforms I and II from mitochondria may be a key regulatory step in apoptosis. Experiments here should reveal the mechanisms of release of hexokinase isoforms from the mitochondrion, and the subunit structure of hexokinase isoforms in their membrane-bound states. Mitochondrial binding and release properties of mutant forms of hexokinase types I and II will test specific mechanisms of ligand-induced release. Studies of spin-labeled hexokinase bound to reconstituted vesicles, using electron paramagnetic resonance methods, will determine whether the enzyme exists as a multimer, and if so, the relative arrangement of subunits in that multimer. The release of hexokinase from the mitochondrion sensitizes cancer cell lines to chemotherapeutic agents. Work on the release mechanisms of hexokinase isoforms I and II, and the structure of the hexokinase membrane-bound state, may reveal new approaches in triggering apoptosis in cancer cells. Research impacts on human health by laying new foundations for the development of drugs in the treatment of diabetes and cancer. Proposed research will reveal strategies for the inhibition of a key enzyme in the biosynthesis of glucose, the consequence of which should be the reduction of harmful levels of serum glucose in the type-2 diabetic. Targeting a second enzyme sensitizes cells to agents that initiate programmed cell- death, in principle allowing the exploitation of new pharmacological synergies in the treatment of cancer. ? ? ?
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