Bacterial motility and gene expression are controlled by a family of phosphorylated response regulators whose activities are modulated by an associated family of protein-histidine kinases. In chemotaxis there are two response regulators, CheY and CheB, that' a residue that is conserved in all homologous response regulator proteins (5). Two additional highly conserved residues, Asp-12 and Asp-13, bind a Mg2+ ion that is essential for phosphorylation (6, 7). CheY, like many other response regulators, has an associated autophosphatase activity; phospho-CheY has a half-life of =10 sec. Phosphatase activity is enhanced by an auxiliary regulatory protein, CheZ (8).The activity of response regulators is controlled by a family of histidine kinases that are autophosphorylated in the presence of ATP (1-3). The phosphotransfer mechanism involves the intermediate formation of a phosphohistidine residue in the kinase. In chemotaxis, the rate of phosphorylation of the histidine residue in the kinase, CheA, is stimulated by membrane chemoreceptor proteins (9, 10). The phosphoryl group is then rapidly transferred to CheY to control motility. Another chemotaxis response regulator, CheB, also accepts phosphoryl groups from CheA. -CheB provides a feedback adaptation mechanism. Phosphorylation of its N-terminal regulatory domain stimulates a C-terminal catalytic activity that modifies the chemoreceptors to attenuate CheA kinase activity (9, 11).Although in many cases a specific kinase has been implicated in the regulation of a given response regulator, considerable cross specificity has also been observed (12-14). The phenotypes of kinase mutants have indicated that response regulators can be phosphorylated in the absence of their cognate kinases (15-20). Here we show that CheY and CheB are enzymes that catalyze their own phosphorylation using low molecular weight phospho-donors. Thus, the enzymology of aspartate phosphorylation is an inherent property of the response regulators that can occur independently of any other protein. Proteins. Wild-type and mutant CheY proteins as well as CheZ and CheB were purified as described (23-25). Protein purity was estimated to be >95% on the basis of SDS/PAGE. MATERIALS AND METHODS Materials[3H]Methyl-labeled Tar receptor in Escherichia coli membranes was prepared as described (25 718The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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 direct 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.

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 ␥-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.

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