Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: Phosphate dependence and effects of other oxyanions

The effects of various oxyanions on the activities of rat liver 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) were examined. No evidence could be found for an absolute dependence of the kinase activity on inorganic phosphate as was recently reported by M. Laloux, E. Van Schaftingen, and H.-G. Hers ((1985) Eur. J. Biochem. 148, 155-159). Rather, phosphate and arsenate activated the kinase by decreasing the enzyme's K_m for fructose 6-phosphate without affecting its K_m for ATP or V_max. The K_m of the kinase for fructose 6-phosphate in the presence of inorganic phosphate was found to be significantly lower (6 μm) than previously reported (30 μm) when the hydrolysis of fructose 2,6-bisphosphate by the concomitant bisphosphatase activity at low Fru 6-P concentrations was taken into account. The K_A's for phosphate and arsenate activation of the kinase were 0.2 and 0.3 mm, respectively. A number of other oxyanions, including pyrophosphate, sulfate, tungstate, selenate, and molybdate all inhibited the kinase by increasing the K_m for fructose 6-phosphate. The apparent K_i's for inhibition of the kinase were in the 0.5-1 mm range. In contrast, all of these oxyanions activated the bisphosphatase, with half-maximal effects requiring millimolar concentrations. Inorganic phosphate was the most potent activator with a K_A of 1 mm. In contrast to the other oxyanions, vanadate and meta-periodate inhibited the kinase but had no effect on the bisphosphatase. Vanadate appeared to be a noncompetitive inhibitor since its effects were not overcome by P_i, ATP, or fructose 6-phosphate, and the species responsible was shown to be decavanadate. Like vanadate, meta-periodate had no effect on the bisphosphatase, though it was a potent inhibitor (I_0.5 = 30 μM) of the kinase. Its effects were shown to be time-dependent and reversed by dithiothreitol, suggesting that it acted by an oxidative mechanism. These results augment the mounting body of evidence that the enzyme's two reactions are catalyzed at discrete active sites.