Activation of a Cyanobacterial Adenylate Cyclase, CyaC, by Autophosphorylation and a Subsequent Phosphotransfer Reaction*-猫眼课题宝

通过自磷酸化和随后的磷酸转移反应激活蓝藻腺苷酸环化酶 CyaC*

DOI：

10.1074/jbc.274.21.15167

发表时间：

1999

期刊：

The Journal of Biological Chemistry

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

M. Kasahara;M. Ohmori

CyaC蛋白是一种蓝藻腺苷酸环化酶，具有独特的一级结构，由腺苷酸环化酶的催化结构域和细菌双组分调控系统的保守结构域组成，即一个发射结构域和两个接收结构域。在本工作中，CyaC作为组氨酸标记的重组蛋白在大肠杆菌中产生并纯化至均一。CyaC在体外可与[γ-32 P]ATP的γ-磷酸发生自磷酸化。CyaC衍生物通过定点诱变构建，其中在发射器结构域（His 572）和接收器结构域（Asp 60或Asp 895）中的高度保守的磷酸化位点分别被谷氨酰胺和丙氨酸残基取代。CyaC衍生物的自磷酸化后，结合到衍生物的磷酰基的化学稳定性进行了测定。发现His 572是初始磷酸化位点，并且一旦与His 572结合，磷酰基就会转移到Asp 895。His 572或Asp 895缺陷的CyaC衍生物的酶活性显著降低。Asp 895被乙酰[32 P]磷酸（一种小的磷酰基分子）磷酸化，但Asp 60不是。乙酰磷酸刺激腺苷酸环化酶的活性，只有当Asp 895是完整的。这些结果表明，Asp 895的磷酸化是必不可少的腺苷酸环化酶的激活和Asp 60的功能不同，从Asp 895在调节酶的活性。

The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced inEscherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylatein vitro with the γ-phosphate of [γ-32P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895. The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced. Asp895 is phosphorylated by acetyl [32P]phosphate, a small phosphoryl molecule, but Asp60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact. These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity.