Cyclic AMP-dependent protein kinase I: Cyclic nucleotide binding, structural changes, and release of the catalytic subunits

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Abstract
Type I cAMP-dependent protein kinase [bovine skeletal muscle] is composed of a dimeric regulatory subunit (R2) and 2 catalytic subnits (C subunits). The R2 dimer binds 4 cAMP molecules to release the 2 C subunits. To characterize the cAMP binding sites and elucidate their role in the release of the C subunits, the R2 dimer was studied by equilibrium methods. The cAMP titration of R2 was monitored by endogenous tryptophan fluorescence, and the results suggest 1 class of binding sites. The titration plot is monotonic for saturation of 4 sites per R2. A similar titration monitored by near-UV circular dichroic changes exhibited profound changes in the region of the 1Lb tyrosine and 1La and 1Lb tryptophan transitions; a plot of these data also showed a linear monotonic response. The fluorescence and circular dichroic changes show that cAMP binding to R2 induces a conformational or structural change. The 1 apparent class of binding sites implies that all binding sites are characterized by similar Kd values or by Kd values much less than the receptor concentration. The reactivity of the cysteine groups with 5,5''-dithiobis(2-nitrobenzoic acid) showed that saturation with cAMP indirectly protects 1 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-cAMP (.epsilon. cAMP), the (.epsilon. cAMP)4.cntdot.R2 complex was titrated with C subunit, causing the release of .epsilon. cAMP. The titration showed that the release of .epsilon. 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 .times. 107 M-1 and 5.0 .times. 108 M-1, respectively. The calculated .DELTA..DELTA.G [change in standard free energy (values for enzymatic inhibitory interactions)] for 1st and last site coupling was 1.9 kcal/mol (1 cal = 4.18 J) of holoenzyme.