8-Substituted cAMP analogues reveal marked differences in adaptability, hydrogen bonding, and charge accommodation between homologous binding sites (AI/AII and BI/BII) in cAMP kinase I and II.
cAMP analogues, systematically substituted at position 8 of the adenine moiety (C8), were tested quantitatively for binding to each cAMP interaction site (A and B) of the regulatory subunits of cAMP-dependent protein kinase type I (RI) and II (RII). Site AII did not accommodate cAMP analogues with any bulk at position 8, whereas site AI accepted even bulky 8-substituents. This implies that the narrow, buried pocket of site AI facing position C8 of cAMP in the RI-cAMP crystal [Su, Y., Dostmann, W. R., Herberg, F. W., Durick, K., Xuong, N. H., Ten Eyck, L., Taylor, S. S., and Varughese, K. I. (1995) Science 269, 807-813] must undergo considerable conformational change and still support high-affinity cAMP analogue binding. The B sites of RI and RII differed in three respects. First, site BI had a lower affinity than site BII for cAMP analogues with hydrophobic, bulky 8-substituents. Second, site BI had a preference for substituents with hydrogen bonding donor potential close to C8, whereas site BII had a preference for substituents with hydrogen bonding acceptor potential. This implies that Tyr(371) of RI and the homologous Tyr(379) of RII differ in their hydrogen bonding preference. Third, site BI preferred analogues with a positively charged amino group that was an extended distance from C8, whereas site BII discriminated against a positive charge. The combined results allow refinement of the cAMP binding site geometry of RI and RII in solution, and suggest design of improved isozyme-specific cAMP analogues.
Centre for Environmental Research and Environmental Technology, Department of Bioorganic Chemistry, University of Bremen, Germany.
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Schwede
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Christensen
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Liauw
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Hippe
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Kopperud
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Jastorff
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Døskeland
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008968:Molecular Conformation; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011817:Rabbits; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007527:Isoenzymes; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases