R. Jakobi et Ja. Traugh, ANALYSIS OF THE ATP GTP BINDING-SITE OF CASEIN KINASE-II BY SITE-DIRECTED MUTAGENESIS/, Physiological chemistry and physics and medical NMR, 27(4), 1995, pp. 293-301
Citations number
23
Categorie Soggetti
Biophysics,Biology,Physiology,"Radiology,Nuclear Medicine & Medical Imaging
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.