Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity

In order to understand better the structural and functional relations betwe en protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, th e catalytic metal and the peptidic substrate, we built a structural model o f Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recent ly solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. T he overall structure of the catalytic subunit is close to the structure sol ved by Niefind et at It comprises two lobes, which move relative to each ot her. The peptide used as substrate is tightly bound to the enzyme, at speci fic locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to th e triphosphate moiety of ATP and a residue sufficiently far from the peptid e that could be mutated so as to modify the specificity of the enzyme. Site -directed mutagenesis was used to replace by charged residues both glycine- 48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site, Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl2 and protein kinase CK2 specific peptide substrat es, The catalytic efficiency of the G48D mutant increased by factors of 4 f or ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low act ivity with ATP and no detectable activity with peptide substrates and was a lso inhibited by magnesium, An increased velocity of ADP release by G48D an d the building of an electrostatic barrier between ATP and the peptidic sub strate in G48K could explain these results. The kinetic properties of the m utant G177K with ATP were not affected, but the catalytic efficiency for th e RRRADDSDDDDD substrate increased sixfold, Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 t owards acidic substrate, thereby increasing its activity.