Surette M G, Levit M, Liu Y, Lukat G, Ninfa E G, Ninfa A, Stock J B
Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
J Biol Chem. 1996 Jan 12;271(2):939-45. doi: 10.1074/jbc.271.2.939.
The histidine protein kinase CheA plays an essential role in stimulus-response coupling during bacterial chemotaxis. The kinase is a homodimer that catalyzes the reversible transfer of a gamma-phosphoryl group from ATP to the N-3 position of one of its own histidine residues. Kinetic studies of rates of autophosphorylation show a second order dependence on CheA concentrations at submicromolar levels that is consistent with dissociation of the homodimer into inactive monomers. The dissociation was confirmed by chemical cross-linking studies. The dissociation constant (CheA2<==>2CheA; KD = 0.2-0.4 microM) was not affected by nucleotide binding, histidine phosphorylation, or binding of the response regulator, CheY. The turnover number per active site within a dimer (assuming 2 independent sites/dimer) at saturating ATP was approximately 10/min. The kinetics of autophosphorylation and ATP/ADP exchange indicated that the dissociation constants of ATP and ADP bound to CheA were similar (KD values approximately 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA approximately P (KD approximately 0.8 mM) compared with ADP (KD approximately 0.3 mM). The rates of phosphotransfer from bound ATP to the phosphoaccepting histidine and from the phosphohistidine back to ADP seem to be essentially equal (kcat approximately 10 min-1).
组氨酸蛋白激酶CheA在细菌趋化性的刺激-反应偶联过程中起着至关重要的作用。该激酶是一种同二聚体，催化γ-磷酸基团从ATP可逆地转移至其自身一个组氨酸残基的N-3位。自磷酸化速率的动力学研究表明，在亚微摩尔水平下，自磷酸化速率对CheA浓度呈二级依赖性，这与同二聚体解离为无活性单体的情况一致。化学交联研究证实了解离现象。解离常数（CheA2<==>2CheA；KD = 0.2 - 0.4 microM）不受核苷酸结合、组氨酸磷酸化或应答调节蛋白CheY结合的影响。在ATP饱和时，二聚体内每个活性位点的周转数（假设每个二聚体有2个独立位点）约为10/分钟。自磷酸化和ATP/ADP交换的动力学表明，与CheA结合的ATP和ADP的解离常数相似（KD值约为0.2 - 0.3 mM），而与ADP（KD约为0.3 mM）相比，ATP与CheA~P的亲和力降低（KD约为0.8 mM）。从结合的ATP向磷酸化接受组氨酸的磷转移速率以及从磷酸组氨酸向ADP的磷转移速率似乎基本相等（kcat约为10 min-1）。
