The long term objective of this proposal is to elucidate regulatory mechanisms involved in carbohydrate metabolism in various mammalian tissues. Interest initially in the regulation of phosphofructokinase, one of the key regulatory enzymes of glycolysis, led in 1980 to the discovery of a very potent naturally occurring activator of phosphofructokinase in liver, fructose 2,6-P2. Subsequently, Fru2,6-P2 was found in all mammalian tissues, in plants, and yeast, thus stimulating a new investigation into the roles and regulation of Fru 2,6-P2 in controlling carbohydrate metabolism. There is strong evidence that this compound plays an important role in dietary and hormonal regulation of glucose homeostasis in liver and is also important in the stimulation of glycolysis in heart muscle under epinephrine and insulin stimulation. There is a strong medical and pharmaceutical importance of understanding the regulation of Fru2,6-P2 in diabetes. Fru 2,6-P2 is synthesized and degraded by a bi functional enzyme, fructose 6-P,2-kinase/fructose 2,6-bisphosphatase in an allosterically controlled fashion. Several tissue-specific isozymic forms of the kinase/phosphatase enzyme (M.W. 55-60 kD) including testes, liver and heart have been isolated that demonstrate different regulatory control mechanisms. The structure of the enzyme from rat testis has recently been solved with data to 2.0 A and has revealed a wealth of information as to the functional architecture of the enzyme and the specific amino acid residues that define the binding and catalytic sites of the enzyme. This structure is an important accomplishment and will serve as a foundation to help guide and interpret experiments focused on dissecting the relationship between structure and function and the allosteric communication between the kinase and phosphatase subunits of this bifunctional enzyme.
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