Interconvertible forms of phosphofructokinase of rabbit liver. The role of effectors on the interconversion.

Abstract

When the phosphofructokinase reaction was started with the addition of stock rabbit liver phosphofructokinase its activity declined rapidly, at low concentrations of fructose 6‐phosphate or at high concentrations of ATP. The rate of this decline is independent of enzyme concentration and it could be prevented by the presence of positive effectors of the enzyme or by high concentration of fructose 6‐phosphate.When the reaction rate was measured in the first few seconds after addition of stock enzyme, the activity of the enzyme and its [S]0.5 value (concentration of fructose 6‐phosphate for half maximal velocity) were not affected significantly by the presence of positive effectors of the enzyme.The stock enzyme, which was routinely stored in the presence of 0.7 M (NH4)2SO4, 10 mM K2HPO4, 10 mM ATP, 50 mM Tris‐HCl pH 7.5 and 0.33 mM EDTA, was transformed to a less active form of enzyme, which had a very high [S]0.5 value and low intrinsic activity on dilution or when freed of effectors by passing the enzyme solution through a Sephadex G‐25 column. This form can be converted back to the active form by the positive effectors of the enzyme. The rate of this conversion of less active form to the active form of enzymes is again independent of enzyme concentration.The rate‐limiting step in the interconversions of active form to less active form of enzyme or vice versa is a first‐order process and there appears to be no aggregation or disaggregation of the enzyme during these interconversions.A variety of [S]0.5 values and maximal velocities were observed for the enzyme depending on the preincubation, prior dilution and removal of the effectors of enzyme suggesting the existence of at least two conformational states of enzyme in varying proportions differing widely in their [S]0.5 values and their intrinsic activities.These results are interpreted in terms of the model of Monod et al. for allosteric enzymes with minor modifications and the hysteretic concept of Frieden.