Studies on Bovine Hepatic Fructose 1,6-Diphosphatase

Abstract

Abstract The kinetics of purified bovine hepatic fructose 1,6-diphosphatase have been examined at physiological pH. The enzyme is inhibited by high concentrations of both its substrate, fructose 1,6-diphosphate (Fru-1,6-P2), and its cofactor magnesium. Magnesium inhibits at noninhibitory Fru-1,6-P2 concentrations, but at inhibitory substrate concentrations, magnesium activates rather than inhibits. Magnesium inhibition is competitive with Fru-1,6-P2 and the Ki (or Kss) for magnesium inhibition is the same as the Kdiss for (MgFru-1,6-P2)-1, indicating that magnesium inhibits by forming a chelate with the free substrate. This suggests that (MgFru-1,6-P2)-1 is not a substrate and the true substrate may be free Fru-1,6-P2. Inhibition by high concentrations of Fru-1,6-P2 is hyperbolic, and noncompetitive with respect to magnesium. The double reciprocal plot of v versus Mg2+ concentration is intersecting, indicating a sequential mechanism for Mg2+ and Fru-1,6-P2 binding. The mechanism is not equilibrium ordered; that is, in the sequential mechanism, either magnesium or substrate can bind to the enzyme first. The evidence for the existence of an allosteric site for substrate inhibition, as well as its possible physiological significance are discussed.