Kinetic Evidence for Multiple Binding Sites on Phosphofructokinase

Abstract

For brain phosphofructokinase at pH 8, with noninhibitory levels of adenosine triphosphate, the Michaelis constants for ATP (0.1 mm) and fructose-6-P (0.04 mm) are each independent of the concentration of the second substrate. Inhibition of P-fructokinase by ATP is highly susceptible to many influences. In addition to the specific effects of NH4+, inorganic orthophosphate, AMP, fructose-6-P, and fructose diphosphate, ATP inhibition is affected by pH, the concentration of Mg2+, dimercaptopropanol, and ethylenediaminetetraacetate. At pH 8, with a high constant level of Mg2+, the velocities can be quantitatively accounted for, over a wide range of levels of both substrates, by a formulation involving two inhibitor sites for ATP and three deinhibitor sites for fructose-6-P, none of which influences the substrate site Michaelis constants for ATP or fructose-6-P. Free ATP is much more inhibitory than MgATP2-. Because of this, and because Mg2+ itself is inhibitory, the relationship between Mg2+ concentration and activity is complex. At pH 8 an Mg2+:ATP ratio of about 2:1 is optimal except at very low ATP levels; at pH 7 much higher Mg2+:ATP ratios are required for maximal rates. Inhibition by Mg2+ is antagonized by AMP. NH4+, Pi, and AMP all increase activity, particularly at inhibitory levels of ATP. The effects are synergistic. At pH 7 with almost complete inhibition by ATP, the results of adding various combinations of the three activators fit a formulation in which the catalytic activity increases progressively as one, two, or three of the activators are bound to the molecule. The formulation requires that the presence of any two of the activators increases the affinity of the enzyme for the third.