Characterization of the calmodulin-binding sites of muscle phosphofructokinase and comparison with known calmodulin-binding domains.
Calmodulin has been shown to interact with high affinity with muscle phosphofructokinase (Mayr, G. W. (1984) Eur. J. Biochem. 143, 513-520, 521-529). In this study, direct binding measurements indicated that each of the two subunits of dimeric phosphofructokinase bound two calmodulins with Kd values of about 3 nM and 1 microM, respectively, in a strictly Ca2+-dependent way. To get more detailed information about this interaction, calmodulin-binding fragments were isolated from a CNBr digest of phosphofructokinase using affinity chromatography on calmodulin-agarose. Two fragments, M11 (Mr 3080) and M22 (Mr 8060), formed a 1:1 stoichiometric complex with Ca2+-calmodulin. The amino acid sequences of these fragments were determined, and their positions in the three-dimensional structure-model of phosphofructokinase are proposed. Fragment M11, which binds to calmodulin with the higher affinity (Kd 11.4 nM), is located in a region of the subunit where two dimers have been proposed to make contacts if associating to active tetrameric enzyme. A stabilization of the dimeric form of the enzyme by binding of calmodulin supports this location of M11. The weaker binding fragment M22 (Kd 198 nM) corresponds to the C-terminal part of the polypeptide and contains the site which is phosphorylated by cAMP-dependent protein kinase. Both fragments have structural properties in common with the isolated calmodulin-binding domains of myosin light chain kinase: two cationic segments rich in hydrophobic residues, one constantly possessing a tryptophan, and the other exhibiting an amino acid sequence resembling sites phosphorylated by cAMP-dependent protein kinase.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits
D000596:Amino Acids; D002147:Calmodulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010732:Phosphofructokinase-1