cAMP-dependent protein kinase (cAPK) is,a heterotetramer containing two regulatory (R) and two catalytic (C) subunits. Each R-subunit contains two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domains, Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activation and to delineate the unique roles that each cAMP-binding domain plays, Two mutations were introduced by replacing the essential Arg in each cAMP-binding site with Lys (R209K in Site A and R333K in Site B), Also, the double mutant (R209/333K) was constructed, Analysis of cAMP binding and dissociation and the apparent constants for holoenzyme activation and R- and C-subunit interaction, measured by analytical gel filtration and surface plasmon resonance, established the following: (1) For rR(R209K), occupancy of Site B is not sufficient to activate the holoenzyme: the low affinity Site A must also be occupied, In rR(R333K), Site A retains its high affinity for cAMP, but Sire A cannot bind until the low affinity Site B is occupied, Thus, both mutants, for different reasons? have similar K-a's for activation that are approximately 20-fold higher than that of the wild-type holoenzyme, The double mutant with two defective sites is no worse than either single mutant. (2) Kinetic analysis of cAMP binding showed that the mutation in Site A or B abolishes high affinity cAMP binding to that site and slightly weakens the affinity of the adjacent site for cAMP. (3) In the presence of MgATP, both mutants rapidly form a stable holoenzyme even in the presence of cAMP in contrast to the wild-type R where holoenzyme forms slowly in vitro and requires dialysis, Regarding the mechanism of activation based on these and other mutants and from kinetic data, the following conclusions are reached: Site A provides the major contact site with the C-subunit, Site B is not essential for holoenzyme formation. Occupancy of Site A by cAMP mediates dissociation of the C-subunit, Site A is inaccessible to cAMP in the full length holoenzyme, while Site B is fully accessible. Access of cAMP to Site A is mediated by Site B, Thus Site B not only helps to shield Sire Al it also provides the specific signal that ''opens up'' Site A, Finally, a nonfunctional Site A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.
