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Jeffry B Stock - One of the best experts on this subject based on the ideXlab platform.
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active site interference and asymmetric activation in the chemotaxis Protein Histidine Kinase chea
Journal of Biological Chemistry, 1996Co-Authors: Mikhail N Levit, Yi Liu, Michael G Surette, Jeffry B StockAbstract:The Histidine Protein Kinase CheA is a multidomain Protein that mediates stimulus-response coupling in bacterial chemotaxis. We have previously shown that the purified Protein exhibits an equilibrium between inactive monomer and active dimer (Surette, M., Levit, M., Liu, Y., Lukat, G., Ninfa, E., Ninfa, A., and Stock, J. (1996) J. Biol. Chem. 271, 939-945). We report here a study of the kinetics of phosphorylation of the isolated phosphoacceptor domain of CheA catalyzed by the isolated catalytic domain of the Protein. The reaction fits Michaelis-Menten kinetics (Km = 0.26 mM for ATP and 0. 10 mM for phosphoacceptor domain; kobs = 17 min-1). The catalytic domain exhibits the same equilibrium between inactive monomers and active dimers as the full-length CheA Protein. Thus, CheA dimerization is an intrinsic property of this domain, independent of any other portion of the molecule and is required for its catalytic activity. In equimolar mixtures of full-length CheA and catalytic domain, homodimers and heterodimers are formed in equal concentration, indicating that all of the determinants for the dimerization are localized entirely on the catalytic domain. An analysis of the kinetics of phosphorylation catalyzed by CheA-catalytic domain heterodimers indicates half of the sites reactivity. The rate of CheA phosphorylation within this heterodimer is over 5-fold greater than that observed in CheA homodimers. The dramatic increase in activity within this asymmetric dimer raises the possibility that CheA activation by receptors involves a mechanism that directs catalysis to one active site while preventing interference from the other.
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dimerization is required for the activity of the Protein Histidine Kinase chea that mediates signal transduction in bacterial chemotaxis
Journal of Biological Chemistry, 1996Co-Authors: Michael G Surette, Mikhail N Levit, Yi Liu, Gudrun Lukat, Elizabeth G Ninfa, Alexander J Ninfa, Jeffry B StockAbstract:Abstract 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 γ-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 μM) 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 ≈ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA∼P (KD ≈ 0.8 mM) compared with ADP (KD≈ 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 ≈ 10 min−).
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dimerization is required for the activity of the Protein Histidine Kinase chea that mediates signal transduction in bacterial chemotaxis
Journal of Biological Chemistry, 1996Co-Authors: Michael G Surette, Mikhail N Levit, Yi Liu, Gudrun Lukat, Elizabeth G Ninfa, Alexander J Ninfa, Jeffry B StockAbstract: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).
Thomas Wieland - One of the best experts on this subject based on the ideXlab platform.
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Nucleoside diphosphate Kinase as Protein Histidine Kinase.
Naunyn-Schmiedeberg's archives of pharmacology, 2014Co-Authors: Paul V. Attwood, Thomas WielandAbstract:Like phosphorylation of serine, threonine, and tyrosine residues in many organisms, reversible Histidine phosphorylation is a well-known regulatory signal in prokaryotes and lower eukaryotes. In vertebrates, phosphoHistidine has been mainly described as a phosphorylated intermediate in enzymatic reactions, and it was believed that regulatory Histidine phosphorylation is of minor importance. During the last decade, it became evident however, that nucleoside diphosphate Kinase (NDPK), an ubiquitously expressed enzyme required for nucleotide homeostasis, can additionally act as a Protein Histidine Kinase. Especially for the isoform NDPK B, at least three defined substrates, the β subunit of heterotrimeric G Proteins (Gβ), the intermediate conductance potassium channel KCa3.1, and the Ca2+-conducting TRP channel family member, TRPV5, have been identified. In all three Proteins, the phosphorylation of a specific Histidine residue is of regulatory importance for Protein function, and these phosphoHistidines are cleaved by a counteracting 14 kDa phosphoHistidine phosphatase (PHP). This article will therefore give an overview of our current knowledge on Protein Histidine phosphorylation in prokaryotes and lower eukaryotes and compare it with the regulatory phosphorylation and dephosphorylation of Histidine residues in vertebrates by NDPK and PHP, respectively.
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reversible Histidine phosphorylation in mammalian cells a teeter totter formed by nucleoside diphosphate Kinase and Protein Histidine phosphatase 1
Methods in Enzymology, 2010Co-Authors: Thomas Wieland, Hansjorg Hippe, Katrin Ludwig, Xiaobo Zhou, Michael Korth, Susanne KlumppAbstract:Regulation of Protein phosphorylation by Kinases and phosphatases is involved in many signaling pathways in mammalian cells. In contrast to prokaryotes and lower eukaryotes a role for the reversible phosphorylation of Histidine residues is just emerging. The β subunit of heterotrimeric G Proteins, the metabolic enzyme adenosine 5'-triphosphate-citrate lyase (ACL), and the Ca2+-activated K+ channel KCa3.1 have been identified as targets for nucleoside diphosphate Kinase (NDPK) acting as Protein Histidine Kinase and the so far only identified mammalian Protein Histidine phosphatase (PHPT-1). Herein, we describe the analysis of the phosphorylation and dephosphorylation of Histidine residues by NDPK and PHPT-1. In addition, experimental protocols for studying the consequences of heterotrimeric G Protein activation via NDPK/Gβγ mediated phosphorelay, the regulation of ACL activity and of KCa3.1 conductivity by Histidine phosphorylation will be presented.
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Interaction of nucleoside diphosphate Kinase B with heterotrimeric G Protein βγ dimers: consequences on G Protein activation and stability
Naunyn-Schmiedeberg's Archives of Pharmacology, 2007Co-Authors: Thomas WielandAbstract:It is generally accepted that G Protein coupled receptors (GPCR) activate heterotrimeric G Proteins by inducing a GDP/GTP exchange at the G Protein α subunit. In addition, the transfer of high energetic phosphate by nucleoside diphosphate Kinase (NDPK) and/or the β subunit of G Proteins (Gβ) can induce G Protein activation. Recent evidence suggests that the NDPK isoform B (NDPK B) forms a complex with Gβγ dimers. In this complex, NDPK B acts as a Protein Histidine Kinase phosphorylating Gβ at Histidine residue 266 (His266). The high energetic phosphoamidate bond on His266 allows for a phosphate transfer specifically onto GDP and thus local formation of GTP, which binds to and thereby activates the respective G Protein α subunit. Apparently, this process occurs independent of the classical GPCR-induced GDP/GTP exchange at least for members of the G_s and G_i subfamilies of heterotrimeric G Proteins. By using a mutant of Gβ_1 in which His266 was replaced by Leu, it was recently demonstrated that NDPK B/Gβγ-mediated G_s activation contributes by about 50% to basal cAMP formation and contractility in rat cardiac myocytes. Besides its apparent role in G Protein activation, the complex formation of NDPK B with Gβγ dimers might be essential for G Protein stability. Depletion of either the NDPK B orthologue or Gβ_1 isoforms in zebrafish embryos led to a similar phenotype displaying contractile dysfunction in the heart accompanied by a complete loss of heterotrimeric G Protein expression. In conclusion, the interaction of NDKP B with Gβγ dimers might play an important role in signal transduction, and alterations in this novel pathway might be of pathophysiological importance.
Michael G Surette - One of the best experts on this subject based on the ideXlab platform.
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active site interference and asymmetric activation in the chemotaxis Protein Histidine Kinase chea
Journal of Biological Chemistry, 1996Co-Authors: Mikhail N Levit, Yi Liu, Michael G Surette, Jeffry B StockAbstract:The Histidine Protein Kinase CheA is a multidomain Protein that mediates stimulus-response coupling in bacterial chemotaxis. We have previously shown that the purified Protein exhibits an equilibrium between inactive monomer and active dimer (Surette, M., Levit, M., Liu, Y., Lukat, G., Ninfa, E., Ninfa, A., and Stock, J. (1996) J. Biol. Chem. 271, 939-945). We report here a study of the kinetics of phosphorylation of the isolated phosphoacceptor domain of CheA catalyzed by the isolated catalytic domain of the Protein. The reaction fits Michaelis-Menten kinetics (Km = 0.26 mM for ATP and 0. 10 mM for phosphoacceptor domain; kobs = 17 min-1). The catalytic domain exhibits the same equilibrium between inactive monomers and active dimers as the full-length CheA Protein. Thus, CheA dimerization is an intrinsic property of this domain, independent of any other portion of the molecule and is required for its catalytic activity. In equimolar mixtures of full-length CheA and catalytic domain, homodimers and heterodimers are formed in equal concentration, indicating that all of the determinants for the dimerization are localized entirely on the catalytic domain. An analysis of the kinetics of phosphorylation catalyzed by CheA-catalytic domain heterodimers indicates half of the sites reactivity. The rate of CheA phosphorylation within this heterodimer is over 5-fold greater than that observed in CheA homodimers. The dramatic increase in activity within this asymmetric dimer raises the possibility that CheA activation by receptors involves a mechanism that directs catalysis to one active site while preventing interference from the other.
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dimerization is required for the activity of the Protein Histidine Kinase chea that mediates signal transduction in bacterial chemotaxis
Journal of Biological Chemistry, 1996Co-Authors: Michael G Surette, Mikhail N Levit, Yi Liu, Gudrun Lukat, Elizabeth G Ninfa, Alexander J Ninfa, Jeffry B StockAbstract:Abstract 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 γ-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 μM) 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 ≈ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA∼P (KD ≈ 0.8 mM) compared with ADP (KD≈ 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 ≈ 10 min−).
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dimerization is required for the activity of the Protein Histidine Kinase chea that mediates signal transduction in bacterial chemotaxis
Journal of Biological Chemistry, 1996Co-Authors: Michael G Surette, Mikhail N Levit, Yi Liu, Gudrun Lukat, Elizabeth G Ninfa, Alexander J Ninfa, Jeffry B StockAbstract: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).
Mikhail N Levit - One of the best experts on this subject based on the ideXlab platform.
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active site interference and asymmetric activation in the chemotaxis Protein Histidine Kinase chea
Journal of Biological Chemistry, 1996Co-Authors: Mikhail N Levit, Yi Liu, Michael G Surette, Jeffry B StockAbstract:The Histidine Protein Kinase CheA is a multidomain Protein that mediates stimulus-response coupling in bacterial chemotaxis. We have previously shown that the purified Protein exhibits an equilibrium between inactive monomer and active dimer (Surette, M., Levit, M., Liu, Y., Lukat, G., Ninfa, E., Ninfa, A., and Stock, J. (1996) J. Biol. Chem. 271, 939-945). We report here a study of the kinetics of phosphorylation of the isolated phosphoacceptor domain of CheA catalyzed by the isolated catalytic domain of the Protein. The reaction fits Michaelis-Menten kinetics (Km = 0.26 mM for ATP and 0. 10 mM for phosphoacceptor domain; kobs = 17 min-1). The catalytic domain exhibits the same equilibrium between inactive monomers and active dimers as the full-length CheA Protein. Thus, CheA dimerization is an intrinsic property of this domain, independent of any other portion of the molecule and is required for its catalytic activity. In equimolar mixtures of full-length CheA and catalytic domain, homodimers and heterodimers are formed in equal concentration, indicating that all of the determinants for the dimerization are localized entirely on the catalytic domain. An analysis of the kinetics of phosphorylation catalyzed by CheA-catalytic domain heterodimers indicates half of the sites reactivity. The rate of CheA phosphorylation within this heterodimer is over 5-fold greater than that observed in CheA homodimers. The dramatic increase in activity within this asymmetric dimer raises the possibility that CheA activation by receptors involves a mechanism that directs catalysis to one active site while preventing interference from the other.
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dimerization is required for the activity of the Protein Histidine Kinase chea that mediates signal transduction in bacterial chemotaxis
Journal of Biological Chemistry, 1996Co-Authors: Michael G Surette, Mikhail N Levit, Yi Liu, Gudrun Lukat, Elizabeth G Ninfa, Alexander J Ninfa, Jeffry B StockAbstract:Abstract 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 γ-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 μM) 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 ≈ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA∼P (KD ≈ 0.8 mM) compared with ADP (KD≈ 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 ≈ 10 min−).
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dimerization is required for the activity of the Protein Histidine Kinase chea that mediates signal transduction in bacterial chemotaxis
Journal of Biological Chemistry, 1996Co-Authors: Michael G Surette, Mikhail N Levit, Yi Liu, Gudrun Lukat, Elizabeth G Ninfa, Alexander J Ninfa, Jeffry B StockAbstract: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).
Yi Liu - One of the best experts on this subject based on the ideXlab platform.
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active site interference and asymmetric activation in the chemotaxis Protein Histidine Kinase chea
Journal of Biological Chemistry, 1996Co-Authors: Mikhail N Levit, Yi Liu, Michael G Surette, Jeffry B StockAbstract:The Histidine Protein Kinase CheA is a multidomain Protein that mediates stimulus-response coupling in bacterial chemotaxis. We have previously shown that the purified Protein exhibits an equilibrium between inactive monomer and active dimer (Surette, M., Levit, M., Liu, Y., Lukat, G., Ninfa, E., Ninfa, A., and Stock, J. (1996) J. Biol. Chem. 271, 939-945). We report here a study of the kinetics of phosphorylation of the isolated phosphoacceptor domain of CheA catalyzed by the isolated catalytic domain of the Protein. The reaction fits Michaelis-Menten kinetics (Km = 0.26 mM for ATP and 0. 10 mM for phosphoacceptor domain; kobs = 17 min-1). The catalytic domain exhibits the same equilibrium between inactive monomers and active dimers as the full-length CheA Protein. Thus, CheA dimerization is an intrinsic property of this domain, independent of any other portion of the molecule and is required for its catalytic activity. In equimolar mixtures of full-length CheA and catalytic domain, homodimers and heterodimers are formed in equal concentration, indicating that all of the determinants for the dimerization are localized entirely on the catalytic domain. An analysis of the kinetics of phosphorylation catalyzed by CheA-catalytic domain heterodimers indicates half of the sites reactivity. The rate of CheA phosphorylation within this heterodimer is over 5-fold greater than that observed in CheA homodimers. The dramatic increase in activity within this asymmetric dimer raises the possibility that CheA activation by receptors involves a mechanism that directs catalysis to one active site while preventing interference from the other.
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dimerization is required for the activity of the Protein Histidine Kinase chea that mediates signal transduction in bacterial chemotaxis
Journal of Biological Chemistry, 1996Co-Authors: Michael G Surette, Mikhail N Levit, Yi Liu, Gudrun Lukat, Elizabeth G Ninfa, Alexander J Ninfa, Jeffry B StockAbstract:Abstract 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 γ-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 μM) 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 ≈ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA∼P (KD ≈ 0.8 mM) compared with ADP (KD≈ 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 ≈ 10 min−).
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dimerization is required for the activity of the Protein Histidine Kinase chea that mediates signal transduction in bacterial chemotaxis
Journal of Biological Chemistry, 1996Co-Authors: Michael G Surette, Mikhail N Levit, Yi Liu, Gudrun Lukat, Elizabeth G Ninfa, Alexander J Ninfa, Jeffry B StockAbstract: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).
