Stewart R C, Jahreis K, Parkinson J S
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
Biochemistry. 2000 Oct 31;39(43):13157-65. doi: 10.1021/bi001100k.
The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (k(cat) approximately 750 s(-)(1)). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His(48)) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA --> CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheADeltaP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheADeltaP2. The CheADeltaP2 reaction rates were significantly slower and the K(m) value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheADeltaP2 (k(cat)/K(m) approximately 1.5 x 10(6) M(-)(1) s(-)(1)) than with wild-type CheA (k(cat)/K(m) approximately 10(8) M(-)(1) s(-)(1)), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (k(cat)/K(m) approximately 5-50 M(-)(1) s(-)(1)). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.
组氨酸蛋白激酶CheA在细菌趋化信号转导途径中起核心作用。自身磷酸化的CheA会非常迅速地（催化常数约为750 s⁻¹）将其磷酸基团传递给CheY。磷酸化的CheY进而影响鞭毛旋转的方向。CheA的自身磷酸化位点（His⁴⁸）位于其N端的P1结构域。相邻的P2结构域为CheY提供了一个高亲和力结合位点，它可能通过将CheY拴系在靠近P1中磷酸供体的位置来促进磷酸转移反应。为了探究P2在大肠杆菌趋化系统中对CheA→CheY磷酸转移反应的作用，我们检测了一种突变型CheA蛋白（CheADeltaP2）的转移动力学，其中98个氨基酸的P2结构域已被一个11个氨基酸的连接子取代。我们使用快速淬灭和停流荧光实验来监测磷酸从磷酸化的野生型CheA和磷酸化的CheADeltaP2转移到CheY的过程。CheADeltaP2的反应速率明显较慢，且米氏常数（Km）值显著高于野生型CheA的相应值。这些结果表明，CheY与CheA的P2结构域结合确实有助于磷酸转移的快速动力学。尽管CheADeltaP2的磷酸转移比野生型CheA慢（催化常数/米氏常数约为1.5×10⁶ M⁻¹ s⁻¹），但仍比磷酸咪唑和其他小分子磷酸供体使CheY磷酸化的动力学快几个数量级（催化常数/米氏常数约为5 - 50 M⁻¹ s⁻¹）。我们得出结论，CheA的P1结构域在趋化信号传导中的磷酸转移速率方面也做出了重要贡献。
