Tyrosine-371 contributes to the positive cooperativity between the two cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I.
The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coli, 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-N3cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371 [Bubis, J., & Taylor, S.S. (1987) Biochemistry 26, 3478-3486]. The site that was targeted for mutagenesis was Tyr-371. The intention was to establish whether the interactions between the tyrosine 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 Kd(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 Ka's for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in Kd(cAMP).(ABSTRACT TRUNCATED AT 250 WORDS)
Bubis
J
J
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Saraswat
L D
LD
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine; D014443:Tyrosine
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D014443:Tyrosine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP