DIVALENT METAL-ION BINDING TO THE CHEY PROTEIN AND ITS SIGNIFICANCE TO PHOSPHOTRANSFER IN BACTERIAL CHEMOTAXIS
DIVALENT METAL-ION BINDING TO THE CHEY PROTEIN AND ITS SIGNIFICANCE TO PHOSPHOTRANSFER IN BACTERIAL CHEMOTAXIS
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DOI：
10.1021/bi00475a004
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发表时间：
1990-06-12
期刊：
影响因子：
2.9
通讯作者：
STOCK, JB
中科院分区：
文献类型：
--
作者：
LUKAT, GS;STOCK, AM;STOCK, JB
细菌趋化性中的信号转导涉及细胞质蛋白 CheA 和 CheY 之间磷酰基的转移。除了 CheA 自磷酸化所需的金属离子之外，二价镁离子也是磷酸盐从 CheA 转移到 CheY 所必需的。这里描述的工作通过荧光研究证明 CheY 含有镁离子结合位点。该位点是促进从磷酸化 CheA 到 CheY 的磷酸转移所需的金属离子位点的有力候选者。镁离子与 CheY 突变体 D13N 的相互作用减弱，缺乏与 D57N 结合的金属离子，以及向这两个突变体的磷酸转移显着减少，与野生型 CheY 的行为形成间接对比。这支持了这样的假设：Asp13 和 Asp57 形成的酸性口袋对于金属结合和磷酸转移活性至关重要。去磷酸化反应也需要金属离子，这增加了常见的金属-磷蛋白过渡态中间体发生磷酸转移和水解反应的可能性。在与 CheY 同源的磷酸化调节蛋白大家族中，所提出的金属离子结合位点和磷酸化位点的高度保守性支持了所有这些蛋白质通过类似催化机制发挥作用的假设。
Signal transduction in bacterial chemotaxis involves transfer of a phosphoryl group between the cytoplasmic proteins CheA and CheY. In addition to the established metal ion requirement for autophosphorylation of CheA, divalent magnesium ions are necessary for the transfer of phosphate from CheA to CheY. The work described here demonstrates via fluorescence studies that CheY contains a magnesium ion binding site. This site is a strong candidate for the metal ion site required to facilitate phosphotransfer from phospho-CheA to CheY. The diminished magnesium ion interaction with CheY mutant D13N and the lack of metal ion binding to D57N along with significant reduction in phosphotransfer to these two mutants are in indirect contrast to the behavior of wild-type CheY. This supports the hypothesis that the acidic pocket formed by Asp13 and Asp57 is essential to metal binding and phosphotransfer activity. Metal ion is also required for the dephosphorylation reaction, raising the possibility that the phosphotransfer and hydrolysis reactions occur by a common metal-phosphoprotein transition-state intermediate. The highly conserved nature of the proposed metal ion binding site and site of phosphorylation within the large family of phosphorylated regulatory proteins that are homologous to CheY supports the hypothesis that all these proteins function by a similar catalytic mechanism.