Skip to main content
OSTI.GOVU.S. Department of Energy Office of Scientific and Technical Information
U.S. Department of Energy
Office of Scientific and Technical Information
 

Tyrosine-371 contributes to the positive cooperativity between the two cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

Journal Article · · Biochemistry; (United States)
DOI:https://doi.org/10.1021/bi00405a026· OSTI ID:5317459
; ;
The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coil, and oligonucleotide-directed mutagenesis was initiated in order to better understand structural changes that are induced as a consequence of cAMP-binding. Photoaffinity labeling of the type I holoenzyme with 8-azidoadenosine 3', 5'-monophosphate (8-N/sub 3/cAMP) leads to covalent modification of two residues, Trp-260 and Trp-371. The site that was targeted for mutagenesis was Tyr-371. The intention was to establish whether the interactions between the tryosine ring and the adenine ring of cAMP are primarily hydrophobic in nature or whether the hydroxyl group is critical for cAMP binding and/or for inducing conformational changes. A single base change converted Tyr-371 to Phe. This yielded an R subunit that reassociated with the catalytic subunit to form holoenzyme and bound 2 mol of cAMP/mol of R monomer. The cAMP binding properties of the holoenzyme that was formed with this mutant R subunit, however, were altered: (a) the apparent K/sub d/(cAMP) was shifted from 16 to 60 nM; (b) Scatchard plots showed no cooperativity between the cAMP binding sites in the mutant in contrast to the positive cooperativity that is observed for the wild-type holoenzyme; (c) the Hill coefficient of 1.6 for the wild-type holoenzyme was reduced to 0.99. The K/sub a/'s for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in K/sub d/(cAMP). Finally, photolabeling of the mutant holoenzyme and of the R/sub 2/ dimer with 8-N/sub 3/(/sup 32/P)cAMP led to the covalent modification of only Trp-260; photolabeling of the second cAMP binding domain was abolished.
Research Organization:
Univ. of California, San Diego (USA)
OSTI ID:
5317459
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:5; ISSN BICHA
Country of Publication:
United States
Language:
English

Similar Records

Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I
Journal Article · Tue Jun 16 00:00:00 EDT 1987 · Biochemistry; (United States) · OSTI ID:6025135
Site-specific mutations in the regulatory subunit of cAMP-dependent protein kinase: selective screening for functional cAMP binding sites. [Escherichia coli]
Conference · Fri May 01 00:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) · OSTI ID:6078777
Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli
Conference · Thu May 01 00:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) · OSTI ID:7108505

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AMP
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CHROMATOGRAPHY
CONFORMATIONAL CHANGES
DAYS LIVING RADIOISOTOPES
ENZYMES
ESCHERICHIA COLI
HYDROXY ACIDS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MICROORGANISMS
MUTAGENESIS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
RADIOISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
TRANSFERASES
TRITIUM COMPOUNDS
TYROSINE