Studies on the function of the two different intrachain cyclic AMP binding sites of heart protein kinase

The regulatory subunit of bovine heart isozyme II cAMP-dependent protein kinase contains two different intrachain cAMP binding sites which differ in their rates of cyclic nucleotide dissociation and specificity of cyclic nucleotide analogue binding. The dissociation of cAMP from intrachain Site 1 is slow compared with that from Site 2. Cyclic nucleotide analogues with C-8 alterations show a marked relative preference for Site 1, whereas N6-modified analogues select Site 2. Cyclic IMP, which prefers Site 2, activates the partially purified heart protein kinase holoenzyme as efficiently as, or more efficiently than, cAMP itself. Dissociation studies indicate that the binding of subsaturating or saturating concentrations of cAMP to isolated regulatory subunit occurs at both sites, whereas cAMP in low concentrations binds mainly to Site 1 of the holoenzyme and occupies both sites as the cAMP concentration is raised. Cyclic IMP binding to Site 2 is stimulated by the simultaneous binding of 8-Br-cAMP to Site 1, indicating that at least one function of Site 1 is cooperativity. It is concluded that Site 2 occupancy, either alone or in combination with Site 1 occupancy, is responsible for protein kinase activation. The catalytic subunit of the holoenzyme may prevent cAMP binding to Site 2, but this restraint is relieved as Site 1 becomes occupied.