Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

Journal Article · Tue Jun 16 04:00:00 EDT 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00386a035· OSTI ID:6025135

Bubis, J; Taylor, S S

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-N/sub 3/-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-N/sub 3/-(/sup 3/H)cAMP with an excess of N/sup 6/-monobutyryl-cAMP nearly abolished the incorporation of 8-N/sub 3/-cAMP into Trp-260, whereas the modification of Tyr-371 was reduced by 49%. When 8-N/sub 3/-(/sup 32/P)cAMP was bound under equilibrium conditions in the presence of various cAMP analogues, N/sup 6/-monobutyryl-cAMP also selectively abolished incorporation of radioactivity into Trp-260, whereas 8-(methylamino)-cAMP preferentially reduced the covalent modification of Tyr-370. Photolabeling with trace amounts of 8-N/sub 3/-(/sup 32/P)cAMP in the presence of saturating amounts of N/sup 6/-monobutyryl-cAMP led to the covalent modification of only Tyr-371. 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. Thus, Trp-260, although it lies at the boundary between domain A and domain B, must be in close contact with the cyclic nucleotide that is bound to domain A. The results also establish unambiguously that N/sup 6/-substituted analogues of cAMP which are selective for the fast dissociation site preferentially bind to the first cAMP binding site in the linear sequence (site A), whereas C-8-substituted analogues which are selective for the slow dissociation site preferentially bind to the second site (site B).

• Research Organization: Univ. of California, San Diego, La Jolla
• OSTI ID: 6025135
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:12; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CHROMATOGRAPHY
CONFIGURATION INTERACTION
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
ENZYMES
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
RADIOISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
TRANSFERASES
TRITIUM COMPOUNDS