Fructose-2,6-Bisphosphate in Control of Hepatic Gluconeogenesis: From Metabolites to Molecular Genetics

Hormonal regulation of hepatic gluconeogenic pathway flux is brought about by phosphorylation/dephosphorylation and control of gene expression of several key regulatory enzymes. Regulation by cAMP dependent phosphorylation occurs at the level of pyruvate kinase and 6-phosphofructo-2-kinase (6PF-1-K)/fructose-2,6-bisphosphatase (Fru-2,6-P₂ase). The latter is a unique bifunctional enzyme that catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P₂), which is an activator of 6PF-1-K and an inhibitor of Fru-1,6-P₂ase. The bifunctional enzyme is a homodimer whose activities are regulated by cAMP dependent protein kinase-catalyzed phosphorylation at a single NH₂-terminal seryl residue/subunit, which results in activation of the Fru-2,6-P₂ase and inhibition of the PF-1-K reactions. Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of Fru-2,6-P₂ levels. 6PF-2-K/Fru-2,6-P₂ase thus provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver. Pyruvate kinase is regulated by both phosphorylation and allosteric effectors. Fru-1,6-P₂, an allosteric activator, also inhibits cAMP-dependent enzyme phosphorylation, and its steady-state concentration is indirectly determined by the level of Fru-2,6-P₂. Therefore, acute regulation of both pyruvate kinase and the bifunctional enzyme provide coordinated control at both the pyruvate/phosphoenolpyruvate and Fru-6-P/Fru-1,6-P₂ substrate cycles. The Fru-2,6-P₂ system is also subject to complex multihormonal long-term control through regulation of 6 PF-2-K/Fru-2,6-P₂ase gene expression. Glucocorticoids are the major factor in turning on this gene in liver, but insulin is also a positive effector. cAMP prevents the effects of glucocorticoids and insulin. Although Fru-2,6-P₂ plays a key role in the regulation of carbon flux in the gluconeogenic pathway, the regulation of this flux depends on several factors and regulation of other key enzymes whose importance varies depending on the dietary and hormonal status of the animal. Molecular cloning of the cDNA encoding PF-2-K/Fru-2,6-P₂ase has elucidated its structure and permitted analysis of its evolutionary origin as well as its tissue distribution and control of its gene expression. The rat liver and skeletal muscle isoforms arose by alternative splicing of a single gene. The muscle form differs from the liver form only at the NH₂-terminal and does not have a cAMP dependent protein kinase phosphorylation site. The hepatic enzyme subunit consists of 470 amino acids. The NH₂-terminal half of the subunit contains the 6PF-2-K domain (residues 1–250), and the COOH-terminal half contains the Fru-2,6-P₂ase domain (residues 251–470). The bisphosphatase reaction is catalyzed via a phosphohistidine enzyme intermediate. This Fru-2,6-P₂ase domain is evolutionarily related to the phosphoglycerate mutase family of enzymes, which also utilize phosphohistidine in their reaction pathway. The 6PF-2-K domain contains a nucleotide binding fold analogous to that found in bacterial 6PF-1-K. On the basis of these findings, the bifunctional enzyme was formed by a gene fusion event involving two glycolytic enzyme catalytic units, i.e., phosphoglycerate mutase and 6PF-1-K. The importance of this system in the regulation of carbohydrate metabolism in many cell types makes it an ideal system to use in molecular approaches to the definitive study of pathway flux and rate-controlling enzymes.