title: Comparison of the two classes of binding sites (A and B) of type I and type II cyclic-AMP-dependent protein kinases by using cyclic nucleotide analogs.
abstract: cAMP analogs, all 96 of which were modified in the adenine moiety, were examined quantitatively for their ability to inhibit the binding of [3H]cAMP to each of the two classes (A and B) of cAMP-binding sites of type I (rabbit skeletal muscle) and type II (bovine heart) cAMP-dependent protein kinase. The study showed that analogs can be constructed that have a higher affinity than cAMP for a binding site. N6-phenyl-cAMP had 18-fold increased affinity for site A of RI (AI) and 40-fold increased affinity for site AII. 2-chloro-8-methylamino-cAMP had a 7-fold increased affinity for BI, and 8-(4-chlorophenylthio)-cAMP had 17-fold increased affinity for BII. Analogs could discriminate between the two classes of binding sites by more than two orders of magnitude in binding affinity: 2-chloro-8-methylamino-cAMP had 170-fold higher affinity for BI than for AI, and 2-n-butyl-8-thiobenzyl-cAMP had 700-fold higher affinity for BII than for AII. Analogs could also discriminate between the homologous binding sites of the isozymes: 2-n-butyl-8-bromo-cAMP had 260-fold higher affinity for AI than for AII (22-fold higher for BII than BI), and 8-piperidino-cAMP had 50-fold higher affinity for BII than for BI (and 50-fold higher for AI than for AII). The data suggest the following conclusions. (a) Stacking interactions are important for the binding of cAMP to all the binding sites. (b) Subtle differences exist between the sites as to the optimal electron distribution in the adenine ring since modifications that withdraw electrons at C2 and donate at C8 favour binding to BI, and disfavour binding to AI and AII. (c) There are no hydrogen bonds between the adenine ring of cAMP and any of the binding sites. (d) All sites bind cAMP in the syn conformation. (e) The subsites adjacent to the N6 and C8 positions may have nonpolar neighbouring regions since hydrophobic substituents at N6 could increase the affinity for AI and AII and similar substituents at C8 could increase the affinity for BII. Finally, (f) the sites differed in their ability to accomodate bulky substituents at C2 and C8. For all compounds tested, their potency as activators of protein kinases I and II was found to correlate, in a predictable fashion, to their mean affinity for the two classes of binding sites, rather than to the affinity for only one of the sites.
authors.affiliation: Cell Biology Research Group, Institute of Anatomy, University of Bergen, Norway.
authors.forename: D
authors.initials: D
authors.lastname: Ogreid
authors.forename: R
authors.initials: R
authors.lastname: Ekanger
authors.forename: R H
authors.initials: RH
authors.lastname: Suva
authors.forename: J P
authors.initials: JP
authors.lastname: Miller
authors.forename: S O
authors.initials: SO
authors.lastname: Døskeland
mesh_terms: D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D013329:Structure-Activity Relationship
chemical_list: D007527:Isoenzymes; D000242:Cyclic AMP; D011494:Protein Kinases