Autophosphorylation of cardiac 3',5'-cyclic AMP-stimulated protein kinase. Kinetic evidence for the regulatory subunit directly acting at the active site in the R2C2 complex.

The mechanism of the autophosphorylation reaction of bovine heart cyclic AMP-dependent protein kinase (ATP: protein phosphotranferase, EC 2.7.1.31) has been further examined using a kinetic approach. The reaction is first order in both ATP and protein kinase when both are present at comparable concentrations. Dilution has no effect on the fraction of regulatory subunit phosphorylated over a given interval of time, and this finding is in accord with the autophosphorylation proceeding via an intramolecular (or, more appropriately in this case, by an intracomplex) reaction. The possibility of regulatory subunit phosphorylation by uncomplex catalytic subunit or another R2C2 complex (the protein kinase complex of two catalytic subunits and the regulatory dimer) was clearly eliminated. These results are compatible with a subunit geometry permitting the regulatory subunit to bind at the protein substrate region of the kinase's active site and to undergo subsequent phosphorylation.