On the Mechanism of Inhibition of Neutral Liver Fructose 1,6‐Bisphosphatase by Fructose 2,6‐Bisphosphate

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Abstract
The inhibitory effect of fructose 2,6-biphosphate on [rat] fructose 1,6-bisphosphatase was reinvestigated in order to solve the apparent contradiction between competition with the substrate and the synergism with AMP, a strictly noncompetitive inhibitor. The effect of fructose 2,6-bisphosphate was compared to that of other ligands of the enyzme, which, like the substrate and methyl (.alpha. + .beta.)fructofuranoside 1,6-bisphosphate bind to the active site or which, like AMP, bind to an allosteric site. An increase in temperature or pH, or the presence of sulfosalicylate, Li or higher concentrations of Mg as well as partial proteolysis by subtilisin increased [I]0.5 [median inhibitory concentration] for fructose 2,6-bisphosphate and AMP without affecting Km. With the exception of the pH change, all these conditions were also without effect on the affinity of the enzyme for the competitive inhibitor, methyl (.alpha. + .beta.)fructofuranoside 1,6-bisphosphate. These observations can be explained by assuming that fructose 2,6-bisphosphate has no affinity for the active site of fructose 1,6-bisphosphatase but binds to an allosteric site which is different from the AMP site. Fructose 2,6-bisphosphate is therefore classified as an allosteric competitive inhibitor and a model is proposed which explains its synergism with AMP as well as the various cooperative effects.