Conversion of allosteric inhibition to activation in phosphofructokinase by protein engineering.

Many enzymes are subject to allosteric control, often with inhibitors and activators binding to the same effector site. Phosphofructokinase in Escherichia coli is such an enzyme, being inhibited by phosphoenolpyruvate (PEP) and activated by ADP and GDP. How do individual interactions with effectors affect the balance between activation and inhibition, especially when both ligands share aspects of the same binding site? We find that mutation of a single residue in the effector site, Glu----Ala 187, leads to PEP being an activator rather than an inhibitor. With low concentrations of the substrate fructose-6-phosphate, the mutant enzyme is more than one hundred times more active than wild-type enzyme at millimolar concentrations of PEP. The classical Monod-Wyman-Changeux two-state model is too simple to account for the properties of the mutant enzyme.