Binding of ATP to the fructose-2,6-bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase leads to activation of its 6-phosphofructo-2-kinase.
To understand the mechanism by which the activity of the 6-phosphofructo-2-kinase (6PF-2K) of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is stimulated by its substrate ATP, we studied two mutants of the enzyme. Mutation of either Arg-279, the penultimate basic residue within the Walker A nucleotide-binding fold in the bisphosphatase domain, or Arg-359 to Ala eliminated the activation of the chicken 6PF-2K by ATP. Binding analysis by fluorescence spectroscopy using 2'(3')-O-(N-methylanthraniloyl)-ATP revealed that the kinase domains of these two mutants, unlike that of the wild type enzyme, showed no cooperativity in ATP binding and that the mutant enzymes possess only the high affinity ATP binding site, suggesting that the ATP binding site on the bisphosphatase domain represents the low affinity site. This conclusion was supported by the result that the affinity of ATP for the isolated bisphosphatase domain is similar to that for the low affinity site in the wild type enzyme. In addition, we found that the 6PF-2K of a chimeric enzyme, in which the last 25 residues of chicken enzyme were replaced with those of the rat enzyme, could not be activated by ATP, despite the fact that the ATP-binding properties of this chimeric enzyme were not different from those of the wild type chicken enzyme. These results demonstrate that activation of the chicken 6PF-2K by ATP may result from allosteric binding of ATP to the bisphosphatase domain where residues Arg-279 and Arg-359 are critically involved and require specific C-terminal sequences.
Yang
Q H
QH
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Zhu
Z
Z
Dong
M Q
MQ
Ling
S
S
Wu
C L
CL
Li
L
L
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002645:Chickens; D002942:Circular Dichroism; D019281:Dimerization; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000378:metabolism
D005456:Fluorescent Dyes; D062367:ortho-Aminobenzoates; C066977:2'-O-(N-methylanthraniloyl)ATP; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases