Kinetic Evidence for Multiple Binding Sites on Phosphofructokinase

Abstract

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. ADP and cyclic 3',5'-AMP appear to act in the same manner as AMP, presumably at the same site. K+ appears to act in the same manner as NH4+ but does not increase enzyme activity to the same degree. At pH 8 and 27°, P-fructokinase loses half its activity in 4 sec and 95% in 2 min. It can be protected by very low levels of either substrate or either product or by AMP or sulfate. The half-life is doubled by 3 x 10-10 m fructose diphosphate. The kinetic findings have been interpreted to indicate the presence of not less than seven substrate, inhibitor, and deinhibitor sites (and possibly as many as 12). It would appear that these sites are so arranged that the addition of one inhibitor (ATP) makes it easier to add the other (citrate) and harder to add the deinhibitors, whereas addition of any two deinhibitors makes it easier to add the third and harder to add either inhibitor.