Ringheim G E, Taylor S S
Department of Chemistry, University of California, San Diego, La Jolla 92093-0654.
J Biol Chem. 1990 Nov 15;265(32):19472-8.
Each protomer of the regulatory subunit dimer of cAMP-dependent protein kinase contains two tandem and homologous cAMP-binding domains, A and B, and cooperative cAMP binding to these two sites promotes holoenzyme dissociation. Several amino acid residues in the type I regulatory subunit, predicted to lie in close proximity to each bound cyclic nucleotide based on affinity labeling and model building, were replaced using recombinant techniques. The mutations included replacement of 1) Glu-200, predicted to hydrogen bond to the 2'-OH of cAMP bound to site A, with Asp, 2) Tyr-371, the site of affinity labeling with 8-N3-cAMP in site B, with Trp, and 3) Phe-247, the position in site A that is homologous to Tyr-371 in site B, with Tyr. Each mutation caused an approximate 2-fold increase in both the Ka(cAMP) and Kd(cAMP); however, the off-rates for cAMP and the characteristic pattern of affinity labeling with 8-N3-cAMP differed markedly for each mutant protein. Furthermore, these mutations affect the cAMP binding properties not only of the site containing the mutation, but of the adjacent nonmutated site as well, thus confirming that extensive cross-communication occurs between the two cAMP-binding domains. Photoaffinity labeling of the native R-subunit results in the covalent modification of two residues, Trp-260 and Tyr-371, by 8-N3-cAMP bound to sites A and B, respectively, with a stoichiometry of 1 mol of 8-N3-cAMP incorporated per mol of R-monomer (Bubis, J., and Taylor, S. S. (1987) Biochemistry 26, 3478-3486). A stoichiometry of 1 mol of 8-N3-cAMP incorporated per R-monomer was observed for each mutant regulatory subunit as well, even when 2 mol of 8-N3-cAMP were bound per R-monomer; however, the major sites of covalent modification were altered as follows: R(Y371/W), Trp-371; R(E200/D), Tyr-371, and R(F247/Y), Tyr-371.
环磷酸腺苷（cAMP）依赖性蛋白激酶调节亚基二聚体的每个原聚体都包含两个串联且同源的cAMP结合结构域，A和B，cAMP与这两个位点的协同结合促进全酶解离。基于亲和标记和模型构建预测，I型调节亚基中的几个氨基酸残基与每个结合的环核苷酸紧密相邻，利用重组技术对其进行了替换。这些突变包括：1）将预测与结合在位点A的cAMP的2'-OH形成氢键的Glu-200替换为Asp；2）将位点B中用8-N3-cAMP进行亲和标记的位点Tyr-371替换为Trp；3）将位点A中与位点B的Tyr-371同源的位置Phe-247替换为Tyr。每个突变都使Ka（cAMP）和Kd（cAMP）增加了约2倍；然而，每个突变蛋白的cAMP解离速率和8-N3-cAMP亲和标记的特征模式明显不同。此外，这些突变不仅影响含突变位点的cAMP结合特性，还影响相邻未突变位点的cAMP结合特性，从而证实两个cAMP结合结构域之间存在广泛的交叉通讯。天然R亚基的光亲和标记导致Trp-260和Tyr-371这两个残基分别被结合在位点A和B的8-N3-cAMP共价修饰，化学计量比为每摩尔R单体掺入1摩尔8-N3-cAMP（布比斯，J.，和泰勒，S.S.（1987年）《生物化学》26，3478 - 3486）。每个突变调节亚基也观察到每R单体掺入1摩尔8-N3-cAMP的化学计量比，即使每R单体结合2摩尔8-N3-cAMP；然而共价修饰的主要位点发生了如下改变：R（Y371/W），Trp-371；R（E200/D），Tyr-371，以及R（F247/Y），Tyr-371。
