Bubis J, Neitzel J J, Saraswat L D, Taylor S S
Department of Chemistry, University of California, San Diego, La Jolla 92093.
J Biol Chem. 1988 Jul 15;263(20):9668-73.
Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.
环磷酸腺苷（cAMP）依赖性蛋白激酶的每个调节亚基在羧基末端都有两个串联的cAMP结合位点，即A位点和B位点。基于与大肠杆菌分解代谢物基因激活蛋白的cAMP结合结构域的序列同源性，为每个cAMP结合结构域构建了一个模型。每个cAMP结合位点的两个保守特征是一个精氨酸和一个谷氨酸，它们分别与带负电荷的磷酸基团以及核糖环上的2'-羟基相互作用。在I型调节亚基中，cAMP结合位点A中的这个精氨酸是Arg-209。已使用重组DNA技术将该精氨酸替换为赖氨酸。所得蛋白质以高亲和力结合cAMP，并与催化亚基结合形成全酶。突变型全酶也被cAMP激活。然而，突变型R亚基每个R单体仅结合1摩尔的cAMP。光亲和标记证实突变型R亚基只有一个功能性的cAMP结合位点。与在Trp-260和Tyr-371处被8-N3cAMP标记的天然R亚基不同，突变型R亚基仅在一个位点Tyr-371处发生共价修饰，该位点与功能性的cAMP结合位点B相关。通过用对位点A或位点B具有高特异性的cAMP类似物激活突变型全酶，也证实了缺乏功能性的cAMP结合位点A。优先结合位点B的8-NH2-甲基cAMP在激活突变型和野生型全酶的能力方面与cAMP相似，而N6-单丁酰cAMP是一种位点A特异性类似物，是突变型全酶的非常差的激活剂。这些结果支持以下结论：1）Arg-209对于cAMP结合位点A至关重要；2）cAMP结合结构域A对于突变型全酶的解离不是必需的。
