The proposed research focuses on enzymes that play central roles in the regulation of glycolysis and gluconeogenesis. Metabolite flux through the glycolytic and gluconeogenic pathways in liver determines the level of serum glucose. Hence, a thorough understanding of regulatory phenomena associated with glycolysis and gluconeogenesis should facilitate the development of new therapies in the treatment of diabetes. The goal here is a complete description of the dynamics of ligand interactions and the molecular mechanisms of catalytic/regulatory actions associated with human hexokinase I and porcine fructose-i 6-bisphosphatase. Hexokinase I is broadly distributed amongst tissues, and is the predominant isoform in the brain and the red blood cell. The isoform of fructose-1,6-bisphosphatase under investigation here predominates in liver. An engineered form of hexokinase I will permit the determination by NMR spectroscopy of the equilibrium constant governing its central kinetic complex. Specific mutants at ligand binding sites should reveal key factors governing interactions between hexokinase I and the mitochondrion. A proposed model for the allosteric regulation of catalysis in hexokinase I will be tested by directed mutation. Predicted outcomes of specific mutations will be verified by steady-state kinetics and structure determinations by X-ray diffraction. Novel constructs of fructose-I ,6-bisphosphatase, which combine subunits of different functional properties into a single tetramer, should reveal the basis of cooperative phenomena in ligand binding and kinetics. Stopped-flow fluorescence of an engineered fructose-1 ,6-bisphosphatase should determine whether the enzyme under conditions of kinetics assays exists in an oligomeric state other than that of a tetramer. Molecular models for allosteric inhibition, ligand binding synergism, and catalysis proposed for fructose-1 ,6-bisphosphatase will be tested by directed mutations and follow-up investigations in fluorescence spectroscopy, steady state kinetics, NMR spectroscopy and structure determinations from single crystals. Finally, the significance and physiological relevance of a newly discovered allosteric effector site on fructose-1,6-bisphosphatase will be investigated by techniques of steady-state kinetics, fluorescence spectroscopy, NMR spectroscopy, directed mutation and X-ray crystallography.
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