The rate of recombination of the subunits (RI and C) of cAMP-dependent protein kinase depends on whether one or two cAMP molecules are bound per RI monomer

To probe the functional significance of the two cAMP-binding sites (A and B) on each regulatory subunit (RI) of cAMP-dependent protein kinase I, the dissociation of cAMP was studied from wild type RI liganded on site A, site B, or both sites, in the absence and presence of catalytic subunit (C). C enhanced the dissociation of cAMP from RI monoliganded on site A or B more than from A,B-biliganded RI, the rate difference being several orders of magnitude in the absence of Mg/ATP and about 7-fold in the presence of Mg/ATP. The catalytically active site of C was involved, since substrates or pseudosubstrates completely and competitively inhibited the action of C in the absence or presence of Mg/ATP. There was no evidence that C, by binding to one monomer of the RI dimer, affected the binding of cAMP to the other monomer. Likewise, there was no evidence for stable complexes of C and cAMP bound to the same R monomer. C enhanced the dissociation of cAMP from R subunits mutated in site A (RIGlu200, which is mutant RI in which glycine 200 is replaced by glutamic acid) or site B (RITrp334, which is mutant RI in which arginine 334 is replaced by tryptophan) to the same extent as from wild type RI monoliganded with cAMP. This indicates that the properties of nonmutated cAMP-binding sites in RIGlu200 and RITrp334 are modulated in a normal manner by C. Mutant RI defective in site A (RIGlu200) had the same rate and equilibrium cAMP binding properties as did site B of RI with its A site unoccupied. This means that mutational inactivation of one cAMP-binding site of RI can occur without altering the other intrachain cAMP site. By all criteria tested, therefore, RIGlu200 appears to be a valid model for RI with a vacant or nonoccupiable site A. Cooperativity of cAMP binding to the two cAMP-binding sites (A and B) of RI was observed only in the presence of C, the apparent Hill coefficient of cAMP binding being about 2 in the presence of a constant, high concentration of free C. C did not induce cooperativity of cAMP binding to RIGlu200 but caused a dramatic decrease of the apparent cAMP affinity of RIGlu200 relative to wild type RI.