Studies on Heart Phosphofructokinase

Abstract

Abstract The kinetic behavior of crystalline heart phosphofructokinase was studied under different conditions. At pH 8.2 the enzyme exhibited Michaelis-Menten kinetics with respect to ATP, fructose 6-phosphate, or Mg++. The Michaelis constants for these substrates were determined. At pH 6.9, ATP inhibited the enzyme. The presence of a lag period was demonstrated when the enzyme was incubated with ATP. Analysis of the kinetic data by means of the Hill equation revealed that the apparent order of the reaction with respect to fructose-6-P was more than one. When the ATP concentration was increased, the apparent order of the reaction was also increased, up to a value of 2.5. Activators such as cyclic 3',5'-AMP reduced the apparent Km for fructose-6-P. Cyclic 3',5'-AMP caused a change to first order kinetics in the presence of low ATP concentrations. At higher concentrations of ATP, cyclic 3',5'-AMP caused a significant decrease in the sigmoid shape of the saturation curves for fructose-6-P, with little change in the apparent order of the reaction. A form of phosphofructokinase was isolated in the absence of stabilizers (ATP and fructose-1,6-di-P). It was found to be more sensitive to ATP inhibition, as evidenced by a lower apparent Ki, and gave curves for fructose-6-P which were more sigmoid than those of the native enzyme. Crystalline heart phosphofructokinase (s20,ω = 14.5) could be reversibly dissociated to a subactive protomer with an s20,ω value of 7.5 at pH 6.5. The enzyme could further be dissociated in the presence of 5 m guanidine HCl to smaller subunits with an s20,ω value of 2.75, which had no enzyme activity. The effect of different ligands on the sedimentation behavior of the enzyme at pH 6.5 was investigated. ATP was found to favor the dissociated form of the enzyme (7.5 S). Fructose 1,6-di-P favored the fully active polymerized form of the enzyme. Based on the present results and previous reports, a model for the molecular organization of phosphofructokinase is presented.