[17] Using analogs to study selectivity and cooperativity of cyclic nucleotide binding sites

Publisher Summary This chapter discusses the use of analogs to study selectivity and cooperativity of cyclic nucleotide binding sites. The chapter illustrates that cyclic AMP-dependent protein kinase holoenzyme is activated when cAMP binds to a dimeric regulatory subunit (R), and the active catalytic subunit (C) dissociates from the holoenzyme complex according to the stoichiometry addressed in the chapter. A technique is developed to study the relative selectivity of various cyclic nucleotide analogs for protein kinase binding sites. The results are applied to the obtained in site-selectivity studies to show that the binding of one class of cAMP analogs, which prefers a single intrachain site can greatly stimulate the binding of another class of site-selective analogs to the other intrachain site. This cooperativity in binding of cyclic nucleotides to both sites can be an important mechanism for amplifying the magnitude and rate of response to an in vivo hormonal elevation of intracellular cAMP levels. In addition, the methodology used to demonstrate site selectivity and cooperativity of cyclic nucleotides in binding to protein kinase can be applied to studies of ligand interactions with multiple binding sites of any receptor.