ACTIVITY OF THE E(75)E(76) MUTANT OF THE A SUBUNIT OF CASEIN KINASE-II FROM XENOPUS-LAEVIS

The cDNA gene coding for the a subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation substituted glutamic acids for Lys(75) and Lys(76), changing the charge distribution of a very basic sequence found in the alpha-subunit. Expression of the mutated cDNA in a pT7-7 vector in E. coli yielded an active mutant recombinant protein that was extensively purified. This mutant was not significantly affected in its app. K-m for casein or a model peptide substrate, nor in its interaction with the activating P subunit. Inhibition by quercetin and by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII alpha E(75)E(76) mutant was at least one order of magnitude less sensitive to inhibition by polyanionic inhibitors such as heparin, poly U, copolyglutamic acid:tyrosine (4:1) and 2,3 diphosphoglycerate.