Probing the cyclic nucleotide binding sites of cAMP-dependent protein kinases I and II with analogs of adenosine 3',5'-cyclic phosphorothioates

• Published in: The Journal of biological chemistry 1990-06, Vol.265 (18), p.10484-10491
• Main Authors: DOSTMANN, W. R. G, TAYLOR, S. S, GENIESER, H.-G, JASTORFF, B, DOÊSKELAND, S. O, OÊGREID, D
• Format: Article
• Language: English
• Subjects: adenosine 3'-5'-cyclic phosphorothioate; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Binding, Competitive; Biological and medical sciences; Cattle; cyclic AMP; Cyclic AMP - analogs & derivatives; Cyclic AMP - chemical synthesis; Cyclic AMP - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Isoenzymes - metabolism; Macromolecular Substances; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Muscles - enzymology; Myocardium - enzymology; Protein Conformation; Protein Kinases - metabolism; Rabbits; Structure-Activity Relationship; Thionucleotides - chemical synthesis; Thionucleotides - metabolism; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)86973-3

A set of cAMP analogs were synthesized that combined exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP. The potency of these compounds to inhibit the binding of [3H]cAMP to sites A and B from type I (rabbit skeletal muscle) and type II (bovine myocardium) cAMP-dependent protein kinase was determined quantitatively. On the average, the Sp isomers had a 5-fold lower affinity for site A and a 30-fold lower affinity for site B of isozyme I than their cyclophosphate homolog. The mean reduction in affinities for the equivalent sites of isozyme II were 20- and 4-fold, respectively. The Rp isomers showed a decrease in affinity of approximately 400-fold and 200-fold for site A and B, respectively, of isozyme I, against 200-fold and 45-fold for site A and B of isozyme II. The Sp substitutions therefore increased the relative preference for site A of isozyme I and site B of isozyme II. The Rp substitution, on the other hand, increased the relative preference for site B of both isozymes. These data show that the Rp and Sp substitutions are tolerated differently by the two intrachain sites of isozymes I and II. They also support the hypothesis that it is the axial, and not the previously proposed equatorial oxygen that contributes the negative charge for the ionic interaction with an invariant arginine in all four binding sites. In addition, they demonstrate that combined modifications in the adenine ring and the cyclic phosphate ring of cAMP can enhance the ability to discriminate between site A and B of one isozyme as well as to discriminate between isozyme I and II. Since Rp analogs of cAMP are known to inhibit activation of cAMP-dependent protein kinases, the findings of the present study have implications for the synthesis of analogs having a very high selectivity for isozyme I or II.