de Wit R J, Hoppe J, Stec W J, Baraniak J, Jastorff B
Eur J Biochem. 1982 Feb;122(1):95-9. doi: 10.1111/j.1432-1033.1982.tb05852.x.
cAMP binding to the 'stable' cAMP-binding sites in the regulatory subunit of the cAMP-dependent protein kinase type I was investigated using a set of 18 selected derivatives. All the tested analogues were competitive with [3H]cAMP and inhibitor constants from 12 nM to 20 microM with the free regulatory subunit were determined. The cAMP molecule seemed to be bound by these specific hydrogen bonds to the 5' and 3' oxygen, the 2' hydroxyl, and an ion pair interaction between the negative charge in equatorial position and a positively charged amino acid side chain. The adenine base is rather unspecifically bound with no hydrogen bonds involved. This binding specificity of the 'stable' site is similar to the requirement for dissociation as determined by the activation of the kinase by a respective analogue. This indicates that occupation of the 'stable' sites leads to activation of the protein kinase. The presence of the catalytic subunit reduced the affinity of most analogues. The binding of one derivative with the negative charge fixed in the axial position is not influenced by the addition of the catalytic subunit and ATP. A plausible model for a conformational change during the activation process in the 'stable' site is discussed.
使用一组18种选定的衍生物研究了环磷酸腺苷（cAMP）与I型cAMP依赖性蛋白激酶调节亚基中“稳定”的cAMP结合位点的结合情况。所有测试的类似物均与[3H]cAMP竞争，并测定了与游离调节亚基的抑制常数，范围为12 nM至20 μM。cAMP分子似乎通过这些特定的氢键与5'和3'氧、2'羟基结合，并且在赤道位置的负电荷与带正电荷的氨基酸侧链之间存在离子对相互作用。腺嘌呤碱基的结合相当不特异，不涉及氢键。“稳定”位点的这种结合特异性类似于相应类似物激活激酶所确定的解离要求。这表明占据“稳定”位点会导致蛋白激酶的激活。催化亚基的存在降低了大多数类似物的亲和力。一种轴向位置固定有负电荷的衍生物的结合不受催化亚基和ATP添加的影响。讨论了“稳定”位点激活过程中构象变化的合理模型。
