Ser644 is important for catalytic activity but is not involved in cAMP-dependent phosphorylation of yeast 6-phosphofructo-2-kinase.
To identify the target amino acid for the cAMP-dependent phosphorylation of yeast 6-phosphofructo-2-kinase Ser644 was mutated to Ala. The plasmid-encoded wild-type and mutant enzymes were overexpressed in E. coli TG2 cells and in the yeast strain DFY658. Like the wild-type enzyme, the Ser644-->Ala mutant was phosphorylated in vivo after addition of glucose to yeast cells and in vitro by the catalytic subunit of protein kinase A. The specific activity of the mutant enzyme was 6-fold lower than that of the wild-type yeast 6-phosphofructo-2-kinase, but both enzymes were activated in response to the addition of glucose to yeast cells.
Institute of Biochemistry, University of Leipzig, School of Medicine, Germany.
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D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D004926:Escherichia coli; D005947:Glucose / Q000494:pharmacology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D025481:Phosphofructokinase-2; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine
D011994:Recombinant Proteins; D012694:Serine; D000242:Cyclic AMP; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D005947:Glucose; D000409:Alanine