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 effectively utilize ATP and GTP in the phosphotransferase reaction. Two residues conserved in the ATP binding domain of other protein kinases are unique to the catalytic (alpha) subunit of casein kinase II. Val-66 is present 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 alpha subunit of casein kinase II were changed to the conserved residues via single and double mutations by site-directed mutagenesis. These mutations enhanced the utilization of ATP over GTP by altering the K(m) values of the alpha subunit for ATP and GTP. Following reconstitution of the catalytic subunit with the regulatory (beta) subunit, both the K(m) and Vmax values of the reconstituted alpha 2 beta 2 holoenzyme were altered. Interestingly, the mutations also reduced or eliminated the 4- to 5-fold increase in catalytic activity observed with the holoenzyme over that of the alpha subunit alone. This was 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 suggest a co-evolution of these separate functions.
Jakobi
R
R
Department of Biochemistry, University of California, Riverside, USA.
Traugh
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D047390:Casein Kinase II; D002942:Circular Dichroism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000235:genetics
D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases