Role of Enzyme Interactions in the Regulation of Gluconeogenesis: MODIFICATION OF THE BINDING PROPERTIES OF FRUCTOSE 1,6-DIPHOSPHATASE BY ADENOSINE MONOPHOSPHATE, ADENOSINE TRIPHOSPHATE, AND FRUCTOSE 1,6-DIPHOSPHATE

Abstract

Abstract AMP, ATP, and fructose 1,6-diphosphate were found to cooperatively influence the binding of homogeneous swine kidney fructose 1,6-diphosphatase to phosphoryl groups in cellulose phosphate columns. All three compounds interacted with the enzyme and decreased its affinity for phosphoryl groups in the column. However, AMP and fructose-1,6-P2 exhibited the highest activity. These effects were specific for AMP, ATP and fructose-1,6-P2 since other metabolites, including various nucleotides, fructose-6-P, and glucose-6-P, did not significantly influence the binding properties of the enzyme. Combinations of any two of these compounds decreased the binding of fructose 1,6-diphosphatase much more than the sum of each one alone. In order to correlate these effects with the binding of these metabolites to the enzyme, gel filtration studies were carried out. It was found that the enzyme binds 4 moles each of AMP, ATP, and fructose-1, 6-P2 per mole of enzyme. The Kd for AMP was 9.7 µm, and in the presence of 0.2 mm fructose-1,6-P2 it was decreased to 5.2 µm. The Kd for AMP increased to 18.5 µm in the presence of 0.4 µm ATP. However, the addition of fructose-1, 6-P2 and ATP did not affect the number of AMP molecules bound, which suggested that the binding sites for these compounds might be adjacent but not identical. The Kd for fructose-1, 6-P2 was 8.5 µm, and it decreased to 3.6 µm in the presence of 0.1 mm AMP. It decreased to 7.0 µm in the presence of 0.4 mm ATP. ATP, which was the least effective in altering the binding of fructose 1, 6-diphosphatase to cellulose-phosphate, had a Kd of 18.3 µm. In the presence of 0.2 mm fructose-1, 6-P2 or 0.2 mm AMP, the Kd for ATP increased to 43.7 µm. The binding of these ligands to the enzyme generally correlated with their ability to decrease the binding of fructose 1,6-diphosphatase to phosphoryl groups in cellulose-phosphate columns. Alterations in the binding properties of the enzyme may result from the formation of substrate-enzyme-allosteric effector complexes which cause a change in the conformation of the enzyme and decrease the charge of specific cationic groups on amino acid residues in the AMP and fructose-1,6-P2 binding sites. These effects on the binding properties of fructose 1,6-diphosphatase were observed at very low concentrations of fructose-1, 6-P2, 20 µm. At physiological concentrations of ATP and AMP a variation in the concentration of fructose-1, 6-P2 between 10 and 30 µm could regulate the release or binding of fructose 1, 6-diphosphatase to free phosphoryl groups.