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

Type I cyclic AMP (cAMP)-dependent protein kinase is composed of a dimeric regulatory subunit (R(2)) and two catalytic subunits (C subunits). The R(2) dimer 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 R(2) dimer has been studied by equilibrium methods. The cAMP titration of R(2) was 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 R(2). A similar titration monitored by near-UV circular dichroic changes exhibited profound changes in the region of the (1)L(b) tyrosine and (1)L(a) and (1)L(b) tryptophan transitions; a plot of these data also showed a linear monotonic response. Thus, the fluorescence and circular dichroic changes show that cAMP binding to R(2) induces a conformational or structural change. The one apparent class of binding sites implies that all binding sites are characterized by similar K(d) values or by K(d) values 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,N(6)-etheno-cyclic AMP (epsilon cAMP), the (epsilon cAMP)(4).R(2) 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 K(a1) and K(an) values obtained by extrapolation were 2.1 x 10(7) M(-1) and 5.0 x 10(8) M(-1), respectively. The calculated DeltaDeltaG for first and last site coupling was 1.9 kcal/mol (1 cal = 4.18 J) of holoenzyme.