Dissection of the nucleotide and metal-phosphate binding sites in cAMP-dependent protein kinase
Dissection of the nucleotide and metal-phosphate binding sites in cAMP-dependent protein kinase
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DOI：
10.1021/bi982672w
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发表时间：
1999-05-11
期刊：
影响因子：
2.9
通讯作者：
Taylor, SS
中科院分区：
文献类型：
--
作者：
Herberg, FW;Doyle, ML;Taylor, SS
cAMP依赖性蛋白激酶（cAPK）的催化（C）亚基在作为全酶复合物的一部分时，根据几个标准更稳定。通过测量游离C亚基在存在和不存在核苷酸和/或二价金属离子的情况下的热稳定性，发现与I型全酶相关的大部分稳定作用可归因于核苷酸。进一步剖析了这种增强的稳定性的具体要求：腺苷使C亚基稳定至5 ℃;然而，二价阳离子（即，Mg 2+、Ca 2+和Mn 2+）与腺苷和腺嘌呤结合时不会增加热稳定性（1）。二价阳离子以及ATP和ADP本身没有影响（2）。来自ATP和ADP的增强的稳定性需要二价阳离子。MnATP（12摄氏度）显示出比CaATP（7摄氏度）和MgATP（5摄氏度）强得多的效果（3）。在全酶复合物或蛋白激酶抑制剂/C亚基复合物中，金属/ATP也是增强稳定性所必需的; RI或RII亚基或PKI单独都不能显著稳定C亚基（4）。对于高热稳定性，第二个低亲和力金属结合位点的占据是必要的（5）。从这些结果中，我们得出结论，腺嘌呤部分的工作独立于金属结合位点，稳定的自由C亚基本身。当β-和γ-磷酸存在时，需要二价金属来定位这些磷酸，并且需要两种金属来实现与单独的腺苷相当的热稳定性。含两种金属的配合物最稳定。来自不同的晶体结构的C亚基的几种构象的比较归因于开放和封闭的形式的C亚基，以更少和更多的热稳定酶，分别。
The catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is more stable by several criteria when it is part of a holoenzyme complex. By measuring the thermal stability of the free C subunit in the presence and absence of nucleotides and/or divalent metal ions, it was found that most of the stabilizing effects associated with the type I holoenzyme could be attributed to the nucleotide. The specific requirements for this enhanced stability were further dissected: Adenosine stabilized the C subunit up to 5 degrees C; however, divalent cations (i.e., Mg2+, Ca2+, and Mn2+) do not increase heat stability in combination with adenosine and adenine (1). Divalent cations as well as ATP and ADP have no effect by themselves (2). The enhanced stability derived from both ATP and ADP requires divalent cations. MnATP (12 degrees C) shows a much stronger effect than CaATP (7 degrees C) and MgATP (5 degrees C) (3). In the holoenzyme complex or the protein kinase inhibitor/C subunit complex, metal/ATP is also required for enhanced stability; neither the RI or RII subunits nor PKI alone stabilize the C subunit significantly (4). For high thermal stability, the occupation of the second, low-affinity metal-binding site is necessary (5). From these results, we concluded that the adenine moiety works independently from the metal-binding sites, stabilizing the free C subunit by itself. When the beta- and gamma-phosphates are present, divalent metals are required for positioning these phosphates, and two metals are required to achieve thermostability comparable to adenosine alone. The complex containing two metals is the most stable. A comparison of several conformations of the C subunit derived from different crystal structures is given attributing open and closed forms of the C subunit to less and more thermostable enzymes, respectively.