Site specificity of casein kinase-2 (TS) from rat liver cytosol. A study with model peptide substrates.
The factors determining the site recognition and phosphorylation by rat liver casein kinase-2 (CK-2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: the nature of the phosphorylatable amino acid, if any; its position relative to the critically required acidic residues; the extension and the structure of the acidic cluster. All of them were tested as substrate and/or competitive inhibitors of CK-2, and their kinetic and inhibition constants were determined. The results suggest the following conclusions. Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. In order to carry out its role of structural determinant the critical acidic cluster must be located on the C-terminal side of the target residue, though not necessarily adjacent to it. The affinity for the protein-binding site, as deduced from Km and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N-terminal side. An acidic residue at position +3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser-Glu-Glu-Ala-Glu-Glu having similar Km but negligible Vmax compared to Ser-Glu-Ala-Glu-Glu-Glu and Ser-Glu-Glu-Glu-Ala-Glu. These data provide a rationale for the substrate specificity of CK-2 and will give a helpful insight into the structure of the protein-binding site of this enzyme.
Marin
O
O
Meggio
F
F
Marchiori
F
F
Borin
G
G
Pinna
L A
LA
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D047388:Casein Kinases; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D000596:Amino Acids; D010455:Peptides; D011494:Protein Kinases; D047388:Casein Kinases