Modulation of the Kinetic Properties of Phosphofructokinase by Ammonium Ions

Ammonium ions were shown to be much more efficient than potassium ions in activating rabbit skeletal muscle phosphofructokinase (EC 2.7.1.11). Under experimental conditions simulating physiological ones (pH 7.2, and inhibitory concentrations of ATP), the apparent dissociation constant of the phosphofructokinase-NH 4 + complex was 0.33 m m . This molarity was shown to lie within the physiological concentration of ammonia in several tissues exhibiting pronounced glycolytic activity. The activation of the enzyme by NH 4 + was very specific and rapid. It was observed also when ITP was the phosphate donor, indicating that the activation was not mere deinhibition of ATP. The form of phosphofructokinase predominant in the presence of NH 4 + exhibited in a qualitative fashion the same allosteric characteristics of the enzyme form prevailing in its absence, i.e. sigmoidicity with respect to fructose 6-phosphate, and sensitivity to inhibition by citrate and higher concentrations of ATP. However, NH 4 + (2 m m ) decreased the K m for ATP (from 0.031 m m to 0.013 m m ) and for fructose 6-phosphate (from 0.34 m m to 0.04 m m ), and increased the K i for citrate (from 0.025 m m to 0.055 m m ) and for ATP (from 0.31 m m to 0.48 m m ). It is proposed that the activation of phosphofructokinase by NH 4 + could be a regulatory mechanism which provides the metabolites necessary for ammonia fixation and the maintenance of its concentration at tolerable levels. Moreover, the possibility that the increase in tissue concentrations of NH 4 + under anoxia might be a contributing factor to the "Pasteur effect" is discussed.