The interaction of fructose 2,6-biphosphate and AMP with rat hepatic fructose 1,6-biphosphatase

The binding of the inhibitory ligands fructose 2,6-biphosphate and AMP to rat liver fructose 1,6-biphosphatase has been investigated. 4 mol of fructose-2,6-P₂ and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-biphosphate exhibits negative cooperativity as indicated by K'₁ > K'₂ > K'₃ ≥ K'₄ and a Hill plot, the curvature of which indicates K'₂/K'₁ < 1, K'₃/K'₂ < 1, and K'₄/K'₃ = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K'₁ < K'₂ < K'₃ < K'₄ and an n(H) of 2.05. Fructose 2,6- and fructose 1,6-biphosphates enhance the binding of AMP as indicated by an increase in the intrinsic association constants. At pH 9.2, where fructose 2,6-biphosphate and AMP inhibition of the enzyme are diminished, fructose 2,6-phosphate bind with a lower affinity but in a positively cooperative manner, whereas AMP exhibits half-sites reactivity with only 2 mol of AMP bound per mol of tetramer. Ultraviolet difference spectroscopy confirmed the results of these binding studies. The site at which fructose 2,6-bisphosphate binds to fructose 1,6-bisphosphatase has been identified as the catalytic site on the basis of the following. 1) Fructose 2,6-biphosphate binds with a stoichiometry of 1 mol/mol of monomer; 2) covalent modification of the active site with acetylimidazole inhibits fructose 2,6-biphosphate binding; and 3) α-methyl D-fructofuranoside-1,6-P₂ and β-methyl D-fructofuranoside-1,6-P₂, substrate analogs, block fructose 2,6-biphosphate binding. We propose that fructose 2,6-biphosphate enhances AMP affinity by binding to the active site of the enzyme and bringing about a conformational change which may be similar to that induced by AMP interaction at the allosteric site.