Studies on the Function of the Protein Kinase * T w o Intrachain cAMP Binding Sites

The regulatory subunit (R) of CAMP-dependent protein kinase from bovine heart contains two intrachain CAMP binding sites which differ in their rate of cyclic nucleotide dissociation and specificity of cyclic nucleotide analog binding. Competition studies indicate that different Site 1-directed cyclic nucleotide analogs with 8-position modifications markedly inhibit each other for binding to R but are minimally affected by Site 2directed analogs which contain 6-position modifications. Likewise, Site 2-directed analogs compete effectively only with other Site 2-directed analogs. These studies suggest a high degree of specificity of these nucleotides for a respective binding site. When efficiency is based on moles of active catalytic subunit (C) per mol of bound cyclic nucleotide, [3H]cIMP, which selects Site 2 of R, activates a partially purified protein kinase holoenzyme as efficiently as, or more efficiently than, 13H]cAMP. When using the holoenzyme, but not free R, dissociation of subsaturating or saturating [3H]cAMP shows that low concentrations of this nucleotide bind mainly to Site 1, but occupancy of Site 2 occurs as concentrations are raised. [3H]cIMP dissociation is rapid, but occurs from both intrachain sites following equilibrium binding to R or holoenzyme. [3H]cIMP (44 HM) binding to Site 2 of the holoenzyme is markedly stimulated by simultaneous binding of 8-Br-CAMP (44 HM) or 8-Ns-cAMP (44 m) to Site 1. This stimulation of binding is not observed for isolated R subunit but can be restored by recombining R with purified C subunit. The C subunit inhibits C3H]cIMP binding. Site 2-directed analogs such as N6-aminohexylcarboxymethyl-CAMP do not stimulate 13H]cIMP binding and cause only a slight increase in 8-N3-[32P]cAMP binding to Site 1. These results indicate that one function of cAMP binding to Site 1 is for stimulating binding to Site 2. The C subunit of the holoenzyme complex may prevent [3H]cAMP binding to Site 2, but this restraint is relieved when Site 1 is occupied.