Multiple inhibitor sites for ATP on muscle phosphofructokinase as influenced by a change of pH: A computer analysis of “non-linear” kinetic data

Abstract

Kinetic studies have been made of the pH-dependent inhibition of phosphofructokinase (ATP: d-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) by ATP. Non-linear relationships between the reciprocal of the initial velocity and concentration of ATP at inhibitory levels indicated the multiple existence of ATP inhibitor sites on phosphofructokinase. Since the conventional graphical method of measuring kinetic parameters from the ordinary Michaelis-Menten kinetic data fails to give decisive values for such non-linear kinetics, a computer method for obtaining the most plausible parameters based on statistical considerations was explored by means of a digital computer. Application of this estimation method to the kinetic data obtained at various pH levels from 6.8 to 9.6 led to the following conclusions. (1) Multiplicity of the ATP inhibitor sites, or cooperativity of binding of ATP at the inhibitor sites, was found to be most pronounced at pH 7.3 and was reduced by a shift of pH to either side. The precipitous inhibition of the enzyme caused by a minute increase of ATP concentration at a lower pH may be due to a high cooperativity at the inhibitor sites. (2) Affinity of ATP for the inhibitor sites decreased markedly as the pH was raised from 6.8 to 8.0. This reduction of affinity may play a significant role in the removal of the ATP inhibition of the enzyme by raising the pH. A physiological significance of these strange kinetic properties of phosphofructokinase in the cellular regulation of glycolysis is discussed.