Inhibition of rabbit liver fructose 1,6-bisphosphatase by AMP: Effect of temperature and physiological concentrations of cations and anions

Abstract

The sensitivity of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) to inhibition by AMP is highly dependent on the temperature, the ionic composition of the assay mixture, and whether Mg 2+ or Mn 2+ is present as the activating cation. The sensitivity to AMP is increased on addition of K + and decreased by the addition of Na +. At 38 °C, in the presence of physiological concentrations of K +, Na +, HPO 4 2−, and histidine, and with saturating levels of Mn 2+, the concentration of AMP required for 50% inhibition approaches 100 μ m, which is 5–10 times greater than is required with the usual assay conditions. In the presence of both Mg 2+ and Mn 2+, the AMP inhibition curve is identical to that observed with Mn 2+ alone. In agreement with these effects, the affinity of the enzyme for AMP was found to be decreased in the presence of Mn 2+, which also causes the binding curve to become sigmoidal. The preference for Mn 2+ over Mg 2+ was confirmed by measurement of Mn 2+ binding. The enzyme tetramer was found to bind 8 eq of Mn 2+, 4 with high and 4 with lower affinity. In the presence of the substrate analog (α + β)methyl- d-fructofuranoside the number of binding sites in each set is increased to eight. Mg 2+ competes for the low-affinity binding sites, but not for the sites with high affinity. The results suggest that the enzyme contains eight structural sites for Mn 2+, plus four additional sites that bind the Mn 2-substrate (or Mg 2-substrate) complex.