Kinase activity of EnvZ, an osmoregulatory signal transducing protein of Escherichia coli
Kinase activity of EnvZ, an osmoregulatory signal transducing protein of Escherichia coli
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DOI：
10.1006/abbi.1997.0315
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发表时间：
1997-10-15
影响因子：
3.9
通讯作者：
Kenney, LJ
中科院分区：
文献类型：
--
作者：
Kenney, LJ
EnvZ是存在于大肠杆菌中的一种内膜蛋白，其对于膜敏感是重要的并且是孔蛋白基因调控所需的。EnvZ被细胞内ATP磷酸化，EnvZ-P磷酸化OmpR，OmpR然后结合孔蛋白启动子以调节其表达。过表达的，截短形式的酶，EnvZ 115，被用来表征在体外的激酶反应。使用过滤器结合试验，我们报告的激酶活性，包括200 μ M的ATP的表观亲和力的第一次直接测量。磷酸化反应依赖于MgCl 2，并且磷酸酶具有磷酸组氨酸的预期稳定性;即，它在室温下在碱中稳定，在酸中不太稳定。在含有EnvZ的溶液中加入OmpR和ATP导致OmpR刺激的EnvZ依赖性ATP酶活性，但对钒酸盐不敏感。使用免疫复合物激酶反应研究了EnvZ和孔蛋白表达缺陷的两种突变体的体内激酶活性。有趣的是，位于EnvZ周质结构域的突变表现出与野生型酶相同的激酶活性，而位于磷酸化位点附近的突变显示激酶和磷酸转移酶活性均显著降低。这些数据提供了支持的模型EnvZ组成的独立的传感和激酶结构域。(C)1997年学术出版社。
EnvZ is an inner membrane protein present in Escherichia coli that is important for osmosensing and required for porin gene regulation. EnvZ is phosphorylated by intracellular ATP, and EnvZ-P phosphorylates OmpR, which then binds to the porin promoters to regulate their expression. An overexpressed, truncated form of the enzyme, EnvZ115, was used to characterize the kinase reaction in vitro. Using a filter binding assay, we report the first direct measurements of the kinase activity, including the apparent affinity for ATP of 200 mu M. The phosphorylation reaction is dependent on MgCl2, and the phosphoenzyme has the expected stability of a phosphohistidine; i.e., it is stable in base and less stable in acid at room temperature. The addition of OmpR and ATP to solutions containing EnvZ resulted in an OmpR-stimulated, EnvZ-dependent ATPase activity that was not vanadate-sensitive. The in vivo kinase activity of EnvZ and two mutants that were deficient in porin expression were studied using an immune complex kinase reaction. Interestingly, a mutation located in the periplasmic domain of EnvZ exhibited kinase activity that was identical to that of the wild-type enzyme, while a mutation located close to the phosphorylation site showed a significant decrease in both kinase and phosphotransferase activities. These data provide support for models of EnvZ consisting-of separate sensing and kinase domains. (C) 1997 Academic Press.