Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I.
Each regulatory subunit of the cAMP-dependent protein kinase contains two in-tandem cAMP binding sites. Photolabeling of holoenzyme I with 8-azidoadenosine 3',5'-monophosphate (8-N3-cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371. In order to correlate photolabeling of these two residues with occupancy of each specific cAMP binding site, photolabeling was carried out in the presence of various analogues of cAMP that bind preferentially to one site. Photolabeling of holoenzyme I after dissociation of 60% of 8-N3-[3H]cAMP with an excess of N6-monobutyryl-cAMP nearly abolished the incorporation of 8-N3-cAMP into Trp-260, whereas the modification of Tyr-371 was reduced by 49%. When 8-N3-[32P]cAMP was bound under equilibrium conditions in the presence of various cAMP analogues, N6-monobutyryl-cAMP also selectively abolished incorporation of radioactivity into Trp-260, whereas 8-(methylamino)-cAMP preferentially reduced the covalent modification of Tyr-371. Photolabeling with trace amounts of 8-N3-[32P]cAMP in the presence of saturating amounts of N6-monobutyryl-cAMP led to the covalent modification of only Tyr-371. In addition, photolabeling of Tyr-371 was enhanced synergistically in the presence of N6-monobutyryl-cAMP. MgATP reduced the covalent modification of both Trp-260 and Tyr-371 but showed no selectivity for either site. These studies support a model that correlates photolabeling of Trp-260 with occupancy of cAMP binding site A and photolabeling of Tyr-371 with occupancy of cAMP binding site B.(ABSTRACT TRUNCATED AT 250 WORDS)
Bubis
J
J
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology; D010777:Photochemistry; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases