Cyclic AMP-Dependent Protein Kinase I: Cyclic Nucleotide Binding, Structural Changes, and Release of the Catalytic Subunits

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 78; no. 3; pp. 1591 - 1595
• Main Authors: Smith, Stephen B., White, Hillary D., Siegel, Jeffrey B., Krebs, Edwin G.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01-03-1981; National Acad Sciences
• Subjects: Animals; Binding Sites; Biochemistry; Biological Sciences: Biochemistry; Cattle; Circular Dichroism; Cyclic AMP - metabolism; Cyclic nucleotides; Dichroism; Enzyme Activation; Enzymes; Fluorescence; Kinetics; Macromolecular Substances; Molecules; Monomers; Muscles - metabolism; Protein Binding; Protein Conformation; Protein Kinases - metabolism; Reactivity; Spectrometry, Fluorescence; Titration
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.78.3.1591

Type I cyclic AMP (cAMP)-dependent protein kinase is composed of a dimeric regulatory subunit (R2) and two catalytic subunits (C subunits). The R2dimer binds four cAMP molecules to release the two C subunits. To characterize the cAMP binding sites and elucidate their role in the release of the C subunits, the R2dimer has been studied by equilibrium methods. The cAMP titration of R2was monitored by endogenous tryptophan fluorescence, and the results suggest one class of binding sites. The titration plot is monotonic for saturation of four sites per R2. A similar titration monitored by near-UV circular dichroic changes exhibited profound changes in the region of the1Lbtyrosine and1Laand1Lbtryptophan transitions; a plot of these data also showed a linear monotonic response. Thus, the fluorescence and circular dichroic changes show that cAMP binding to R2induces a conformational or structural change. The one apparent class of binding sites implies that all binding sites are characterized by similar Kdvalues or by Kdvalues much less than the receptor concentration. The reactivity of the cysteine sulfhydryl groups with 5,5′-dithiobis(2-nitrobenzoic acid) showed that saturation with cAMP indirectly protects one sulfhydryl group per R monomer. Analysis of cAMP activation of the holoenzyme, detected by phosphotransferase assays, showed that saturation of both cAMP binding sites per R monomer is necessary to effect the release of the C subunit. By using a fluorescent analog of cAMP, 1,N6-etheno-cyclic AMP (ε cAMP), the (ε cAMP)4· R2complex was titrated with C subunit, causing the release of ε cAMP. The titration showed that the release of ε cAMP was a positive cooperative process; its Hill plot had a slope of 2.6 and the Ka1
and Kan
values obtained by extrapolation were 2.1× 107M-1and 5.0× 108M-1, respectively. The calculated Δ Δ G for first and last site coupling was 1.9 kcal/mol (1 cal = 4.18 J) of holoenzyme.