Mutation of Monofunctional 6-Phosphofructo-2-kinase in Yeast to Bifunctional 6-Phosphofructo-2-kinase/Fructose 2,6-Bisphosphatase

We have shown previously that 6-phosphofructo-2-kinase in yeast has negligible fructose-2,6-bisphosphatase activity even though resembling in part of its C-terminal sequence the phosphatase domain of the bifunctional liver enzyme. Here we show that exchanging Ser-404 to His-404 in the yeast peptide creates a bifunctional enzyme with a fructose-2,6-bisphosphatase activity involving a phosphoprotein intermediate. Like mammalian bifunctional enzymes, the His-404 mutant protein is readily phosphorylated by fructose 2,6-P₂ with a half-saturation of 0.4 µM, the same K_m value as for its fructose-2,6-bisphosphatase activity. Protein phosphorylation by the C-subunit of cAMP-dependent protein kinase, presumably at a C-terminal consensus site, increases the K_m value to 1.5 µM. The newly created fructose-2,6-bisphosphatase is inhibited competitively by its product fructose 6-P with a K_i of 0.6 mM. No effect of the His-404 mutation was found on 6-phosphofructo-2-kinase activity, in line with the mutant yeast enzyme having independent kinase and phosphatase domains, like its mammalian wild-type counterparts. The results would fit with the evolution of the PFK26 gene having involved fusion between kinase and phosphatase genes—as proposed for the mammalian enzyme—but with accompanying or later silencing of the fructose-2,6-bisphosphatase activity.