Chicken Liver Phosphofructokinase: III. KINETICS AND ALLOSTERIC PROPERTIES

Abstract

Abstract Some kinetic and allosteric properties of crystalline chicken liver phosphofructokinase were investigated. Lineweaver-Burk plots with fructose 6-phosphate and ATP as substrates yield a series of apparently parallel lines. The liver enzyme also catalyzes the phosphorylation of fructose-1-P, and the double reciprocal plot with fructose-1-P as the substrate yields intersecting lines. Phosphorylation of both fructose-6-P and fructose-1-P are lost at 4° at equal rate. Of the many sugar phosphates examined, only glucose-6-P, 6-P-gluconate, and ribulose-5-P relieve ATP inhibition of liver phosphofructokinase. These sugar-Ps are competitive inhibitors of fructose-6-P at noninhibitory concentrations of ATP. Isocitrate and citrate inhibit phosphofructokinase, while NADH does not. Arrhenius plots of enzyme activity of both ATP inhibited and unhibited activities show a sharp break at approximately 15°. The Km value for ATP decreases from 0.041 mm to 0.012 mm below the transition temperature, while the Km for fructose-6-P is not affected by different temperature. These results suggest the existence of two different conformational states of liver phosphofructokinase. Fructose 1,6-diphosphatase at high concentration enhances ATP inhibition of phosphofructokinase, but at lower concentration it relieves the inhibition. The maximum inhibition by FDPase is usually obtained at a molar ratio (fructose diphosphatase to phosphofructokinase) of approximately 150. Moreover, fructose diphosphatase protects phosphofructokinase against cold inactivation. Physiological significance of these observations and a possible mechanism for the regulation of phosphofructokinase by fructose diphosphatase are discussed.