Mutations uncover a role for two magnesium ions in the catalytic mechanism of adenylyl cyclase.
Mutations uncover a role for two magnesium ions in the catalytic mechanism of adenylyl cyclase.
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突变揭示了两个镁离子在腺苷酸环化酶催化机制中的作用。
DOI：
10.1074/jbc.273.31.19650
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Taussig,R
中科院分区：
文献类型：
--
作者：
Zimmermann,G;Zhou,D;Taussig,R
最近对腺酰环化酶晶体结构的测定阐明了许多结构特征，这些结构特征决定了酶的调节特性。此外，通过突变技术对腺酰环化酶进行表征，以及确定P-位点抑制剂的结合位点，已经导致了描述ATP结合位点的模型化研究。尽管取得了这些进展，但腺酰环化酶的催化机制仍然不确定，特别是关于镁离子在这一过程中可能扮演的角色。我们已经鉴定了四种突变的哺乳动物腺酰环化酶，它们对金属的依赖性存在缺陷，这使得我们能够进一步表征金属离子在该酶的催化机制中的作用。野生型腺酰环化酶呈现双相的镁离子剂量-反应曲线，其中高亲和力成分表现出协同作用(Hill系数为1.4)。两个突变(C441R和Y442H)显著降低了腺酰环化酶对镁离子的亲和力，而不影响与镁ATP的结合，这表明除了与ATP结合的镁离子外，还需要一种金属。因此，这项研究的结果证明了腺酰环化酶对金属的多重需求，并支持催化所必需的、有别于ATP结合离子的镁离子的存在。我们认为腺酰环化酶采用了类似于DNA聚合酶的催化机制，其中两个关键的镁离子促进了3‘-羟基的亲核攻击和随后的焦磷酸的消除。
The recent determination of the crystal structure of adenylyl cyclase has elucidated many structural features that determine the regulatory properties of the enzyme. In addition, the characterization of adenylyl cyclase by mutagenic techniques and the identification of the binding site for P-site inhibitors have led to modeling studies that describe the ATP-binding site. Despite these advances, the catalytic mechanism of adenylyl cyclase remains uncertain, especially with respect to the role that magnesium ions may play in this process. We have identified four mutant mammalian adenylyl cyclases defective in their metal dependence, allowing us to further characterize the function of metal ions in the catalytic mechanism of this enzyme. The wild-type adenylyl cyclase shows a biphasic Mg2+dose-response curve in which the high-affinity component displays cooperativity (Hill coefficient of 1.4). Two mutations (C441R and Y442H) reduce the affinity of the adenylyl cyclase for Mg2+dramatically without affecting the binding of MgATP, suggesting that there is a metal requirement in addition to the ATP-bound Mg2+. The results of this study thus demonstrate multiple metal requirements of adenylyl cyclase and support the existence of a Mg2+ion essential for catalysis and distinct from the ATP-bound ion. We propose that adenylyl cyclase employs a catalytic mechanism analogous to that of DNA polymerase, in which two key magnesium ions facilitate the nucleophilic attack of the 3′-hydroxyl group and the subsequent elimination of pyrophosphate.