Regulation of human erythrocyte hexokinase: The influence of glycolytic intermediates and inorganic phosphate

Abstract

Human erythrocyte hexokinase (ATP : d-hexose 6-phosphotransferase, EC 2.7.1.1) was inhibited competitively with respect to MgATP 2− by glucose-6- P ( K i = 10.8 μM) and fructose-6- P ( K i = 160 μM). Low concentrations of inorganic phosphate were competitive with respect to glucose-6- P and fructose-6- P, although higher concentrations of P i were not able to overcome completely the inhibition by the hexose phosphates. The results are consistent with a model in which hexokinase exists in equilibrium either as free or phosphate-associated enzyme, the latter having a reduced but still substantial affinity for hexose phosphate. An alternative explanation could be found in the presence of two different enzymes, one with a high affinity for glucose-6- P being sensitive to regulation by P i, one with a lower affinity for glucose-6- P being insensitive to P i. A similar but less pronounced effect of P i, was found on the inhibition by 2,3-diphosphoglycerate ( K i = 4.0 mM). P i in the absence of inhibitor was also a competitive inhibitor with respect to MgATP 2− ( K i = 20 mM). Furthermore a competitive inhibition with respect to MgATP 2− was found by fructose 1,6-diphosphate ( K i = 4.3 mM), glycerate-3- P ( K i = 3.8 mM), glycerate-2- P ( K i = 12.5 mM), MgADP− ( K i = 1.0 mM) and MgAMP ( K i = 1.7 mM).