SITE-DIRECTED MUTAGENESIS AND STRUCTURE-FUNCTION STUDIES OF CASEIN KINASE-II CORRELATE STIMULATION OF ACTIVITY BY THE BETA-SUBUNIT WITH CHANGES IN CONFORMATION AND ATP/GTP UTILIZATION

Casein kinase II exists in vivo as an active holoenzyme consisting of catalytic alpha and/or alpha' and regulatory beta subunits, which form a tetrameric structure of alpha(2) beta(2). Unlike most other protein kinases, casein kinase II uses both ATP and GTP effectively as phosphate donors. Two residues unique to the catalytic subunit of casein kinase II, Val66 and Trp176, were mutated to Ala66 and Phe176, respectively, the amino acids present in more than 95% of the identified protein kinase sequences. Using recombinant alpha subunit expressed in Escherichia coli, the mutations have been previously shown to reduce the utilization of GTP by changing K-m values for ATP and GTP and to reduce the approximate fivefold stimulation observed upon addition of the regulatory subunit [Jakobi, R. and Traugh, J. A. (1992) J. Blob. Chem. 267, 23894-23902]. In this study, the mutations are shown to affect the catalytic activity of the reconstituted holoenzyme by changing both K-m and V-max values. The V-max for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP. The Val66 to alanine and Va166/Trp176 to alanine/phenylalanine mutations reduce the V-max values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the beta subunit are due to changes in V-max. Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of V-max by the beta subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations. Correlation of changes in secondary structure and stimulation of activity by the beta subunit indicate that the formation of the wild-type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction. As shown by the mutations, Val66 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP.