Rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: A unique bifunctional enzyme

Abstract

Fructose 2,6-bisphosphate is a potent allosteric activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. It potentiates the effect of AMP on both enzymes. A great deal of compelling evidence supports the hypothesis that fructose 2,6-bisphosphate plays a key role in the hormonal and substrate regulation of substrate cucling at the fructose 6-phosphate/fructose 1,6-bisphosphate level in liver. This regulaton is exerted at the level of the enzyme activities responsible for the synthesis and degradation of fructose 2,6-bisphosphate. Synthesis of the compound is catalyzed by a unique enzyme which transfers the γ-phosphate of ATP to the C2 position of fructose 6-phosphate (ATP:D fructose 6-phosphate 2-phosphotransferase) while degradation is catalyzed by a phosphohydrolase activity which is specific for the C-2 position of fructose 2,6-bisphosphate (D-fructose 2,6-bisphosphate 2-phosphohydrolase). These activities are distinct from the classical 6-phosphofructo 1-kinase and fructose 1,6-bisphosphatase with regard to molecular weight, interaction with ligands, and the efficiency with which phosphoryl transfer occurs. Both activities have been purified to homogeneity and have been shown to be present in a single enzyme protein, i.e. the enzyme is bifunctional. Incubation of the 6-phosphofructo 2-kinse/fructose 2,6-bisphosphatase with cAMPp-dependent protein kinase and ATP leads to phosphorylation of the enzyme resulting in inactivaton of the phosphotransferase activity and stimulation of the phosphohydrolase activity. Since fructose 2,6-bisphosphate is not further metabolized and can only be recycled to fructose 6-phosphate, simultaneous modulation of a single protein provides a very efficient and sensitive regulatory mechanims. The bifunctional enzyme was also shown to posses an ATPase activity which was nearly equal to the activity of the kinase reaction. However, in the presence of fructose 6-phosphate the enzyme did not transfer phosphate to water but rather to the C-2 position of the phosphorylated sugar. The ability of the enzyme to catalyze a partial reaction at a rate nearly equal to that of the forward reaction suggested that the reaction mechanism of the kinase proceeds by a two step transfer, i.e. via a phosphoryl enzyme intermediate. Additional evidence in support of this was the enzyme's ability to catalyze both ATP-ADP and fructose 6-phosphate-fructose 2,6-bisphosphate exchange reactions and the isolation of an acid labile phosphoryl enzyme intermediate after incubation of the enzyme with [γ- 32P]ATP or [2- 32p]fructose 2,6-bisphosphate. Since most phosphoryl transfer reactions are thought to proceed by a single in line displacement reaction, 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase presents a unique oppurtunity for detailed analyses of the reacton mechanism of a phosphoryl transfer enzyme where covalent catalysis occurs.