The Crystal Complex of Phosphofructokinase-2 of Escherichia coli with Fructose-6-phosphate

Abstract

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6- phosphate (fructose-6-P) and futile consumption of ATP de- laying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 A ˚ . The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative in- terplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K0.5 for fructose-6-P and a decrease in the apparent kcat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (nH of approximately 2). Differences and resem- blances in the patterns of fructose-6-P binding and the mecha- nism of inhibition are discussed for Pfk-1 and Pfk-2, as an ex- ample of evolutionary convergence, because these enzymes do not share a common ancestor.