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 microM, the same Km 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 Km value to 1.5 microM. 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.
Kretschmer
M
M
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Langer
C
C
Prinz
W
W
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006639:Histidine; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D012694:Serine; D013329:Structure-Activity Relationship
D004271:DNA, Fungal; D012694:Serine; D006639:Histidine; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases