Isotope-exchange evidence that glucose 6-phosphate inhibits rat-muscle hexokinase II at an allosteric site.

Abstract

The flux ratio for hexokinase type II from rat muscle, i.e. the rate of conversion of glucose 6-phosphate molecules into ATP molecules divided by the simultaneous rate of conversion of glucose 6-phosphate molecules into glucose molecules, increases with the MgATP concentration but is independent of the glucose concentration. This behaviour requires that glucose must bind before MgATP when the reaction is proceeding in the normal physiological direction, i.e. phosphorylation of glucose. Although at low non-inhibitory glucose 6-phosphate concentrations the flux ratio increases linearly with the MgATP concentration, the dependence becomes non-linear, with a slope that increases with the MgATP concentration, at glucose 6-phosphate concentrations above 1 mM. This behaviour does not permit glucose 6-phosphate to act only as a normal product inhibitor. Instead, it seems to require glucose 6-phosphate to act as an allosteric inhibitor and for a second site for binding of MgATP to exist. Measurements of the flux from ATP to glucose 6-phosphate and to ADP showed no dependence of the flux ratio on the concentrations of either glucose 6-phosphate or ADP. This result does not permit the order of product-release steps in this direction to be determined, but shows that the second product is released virtually instantaneously after first.