The role of fructose 2,6-bisphosphate in regulation of fructose-1,6-bisphosphatase.

The effect of fructose 1,6-bisphosphate and fructose 2,6-bisphosphate on the inhibition of rat liver fructose1,6-bisphosphatase by AMP was investigated. When the concentration of fructose 1,Bbisphosphate increased from 1 p~ to 50 p ~ , the concentration of AMP that gave 50% inhibition (So.6) decreased from 50 PM to 16 p ~ . Fructose 2,6-bisphosphate was also found to potentiate allosteric inhibition of the enzyme by AMP but at much lower concentrations than those required for fructose 1,6-bisphosphate. Thus, both the substrate, fructose 1,6-bisphosphate, and an inhibitor, fructose 2,6-bisphosphateT appear to interact with the allosteric site for AMP. The basis for this interaction was investigated by studying the effect of fructose bisphosphates on modification of the enzyme by acetylation with acetylimidazole. Inclusion of fructose 1,Bbisphosphate during the acetylation reaction protected catalytic activity, but the AMP inhibition was abolished. Inclusion of AMP led to a large loss of catalytic activity but the sensitivity of the residual enzyme activity to AMP inhibition was not altered. However, inclusion of fructose 2,6-bisphosphate protected both the catalytic activity and the sensitivity of the enzyme to AMP inhibition. Ultraviolet difference spectroscopy showed that acetylation occurred at tyrosine residues and that fructose 2,6-bisphosphate or AMP addition to the enzyme induced similar difference spectra. These results suggest that one group of tyrosine residues are associated with the catalytic site and another with the allosteric site and that fructose 2,6-bisphosphate can interact with both groups.