ANALYSIS OF THE ATP GTP BINDING-SITE OF CASEIN KINASE-II BY SITE-DIRECTED MUTAGENESIS/

Casein kinase II is one of only a few protein kinases which effectivel y utilize ATP and GTP in the phosphotransferase reaction. Two residues conserved in the ATP binding domain of other protein kinases are uniq ue to the catalytic (alpha) subunit of casein kinase II. Val-66 is pre sent in subdomain II and Trp-176 in subdomain VII, while > 95% of the other protein kinases contain alanine and phenylalanine, respectively. The residues in the a subunit of casein kinase II were changed to the conserved residues via single and double mutations by site-directed m utagenesis. These mutations enhanced the utilization of ATP over GTP b y altering the K-m values of the alpha subunit for ATP and GTP. Follow ing reconstitution of the catalytic subunit with the regulatory (beta) subunit, both the K-m and V-max values of the reconstituted alpha(2) beta(2) holoenzyme were altered. Interestingly, the mutations also red uced or eliminated the 4- to 5-fold increase in catalytic activity obs erved with the holoenzyme over that of the alpha subunit alone. This w as due to changes in secondary structure of the holoenzyme as shown by UV circular dichroism spectroscopy. Taken together, the data indicate that utilization of both ATP and GTP can be directly correlated with stimulation of catalytic activity by the regulatory subunit and sugges t a co-evolution of these separate functions.