Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate

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Abstract
Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3'',5''-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analog 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound/mol of type I regulatory subunit monomer (RI), the apparent Kd observed were .apprx. 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and the Hill coefficients were .apprx. 1.5-1.6. After photolysis and addition of an excess of cAMP, .apprx. 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that 2 unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the 2 residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue. RI contains 2 adjacent regions of sequence homology in the COOH-terminal fragment that binds 2 molecules of cAMP. On the basis of the homology between the cAMP binding domain of the Escherichia coli catabolite gene activator protein (CAP) and the 2 cAMP-binding domains of RI, a model has been proposed that is consistent with the covalent modifications described above. The modification of proline-271 apparently is due to 8-N3cAMP bound to cAMP binding domain A and modification of tyrosine-371 results from 8-N3cAMP bound to cAMP binding domain B.