Department of Pharmacology, University of California, San Diego, California 92093, USA.
J Am Chem Soc. 2013 Mar 27;135(12):4788-98. doi: 10.1021/ja312237q. Epub 2013 Mar 12.
The catalytic (C) subunit of cAMP-dependent protein kinase (PKA) is a serine/threonine kinase responsible for most of the effects of cAMP signaling, and PKA serves as a prototype for the entire kinase family. Despite multiple studies of PKA, the steps involved in phosphoryl transfer, the roles of the catalytically essential magnesium ions, and the processes that govern the rate-limiting step of ADP release are unresolved. Here we identified conditions that yielded slow phosphoryl transfer of the γ-phosphate from the generally nonhydrolyzable analog of ATP, adenosine-5'-(β,γ-imido)triphosphate (AMP-PNP), onto a substrate peptide within protein crystals. By trapping both products in the crystal lattice, we now have a complete resolution profile of all the catalytic steps. One crystal structure refined to 1.55 Å resolution shows two states of the protein with 55% displaying intact AMP-PNP and an unphosphorylated substrate and 45% displaying transfer of the γ-phosphate of AMP-PNP onto the substrate peptide yielding AMP-PN and a phosphorylated substrate. Another structure refined to 2.15 Å resolution displays complete phosphoryl transfer to the substrate. These structures, in addition to trapping both products in the crystal lattice, implicate one magnesium ion, previously termed Mg2, as the more stably bound ion. Following phosphoryl transfer, Mg2 recruits a water molecule to retain an octahedral coordination geometry suggesting the strong binding character of this magnesium ion, and Mg2 remains in the active site following complete phosphoryl transfer while Mg1 is expelled. Loss of Mg1 may thus be an important part of the rate-limiting step of ADP release.
环腺苷酸（cAMP）依赖性蛋白激酶（PKA）的催化（C）亚基是一种丝氨酸/苏氨酸激酶，负责 cAMP 信号的大部分作用，PKA 作为整个激酶家族的原型。尽管对 PKA 进行了多项研究，但磷酸转移的步骤、催化必需的镁离子的作用以及控制 ADP 释放限速步骤的过程仍未得到解决。在这里，我们确定了条件，使γ-磷酸从通常不可水解的 ATP 类似物腺苷-5'-(β,γ-亚氨基)三磷酸（AMP-PNP）缓慢转移到蛋白质晶体中的底物肽上。通过在晶体晶格中捕获两种产物，我们现在有了所有催化步骤的完整分辨率谱。一个精修至 1.55Å分辨率的晶体结构显示了两种蛋白质状态，其中 55%显示完整的 AMP-PNP 和未磷酸化的底物，45%显示 AMP-PNP 的γ-磷酸转移到底物肽上，生成 AMP-PN 和磷酸化的底物。另一个精修至 2.15Å分辨率的结构显示完全将磷酸转移到底物上。这些结构，除了在晶体晶格中捕获两种产物外，还表明一个镁离子，以前称为 Mg2，是结合更稳定的离子。磷酸转移后，Mg2 招募一个水分子以保持八面体配位几何形状，表明这个镁离子具有很强的结合特性，并且在完全磷酸转移后，Mg2 留在活性位点，而 Mg1 被排出。因此，Mg1 的丢失可能是 ADP 释放限速步骤的重要组成部分。
