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(Received for publication, August 10,
1995; and in revised form, October 11, 1995) From the
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
The histidine protein kinase CheA is a central component of the
system that mediates receptor signaling in bacterial chemotaxis (for
reviews see (1) and (2) ). The protein purified from Escherichia coli or Salmonella typhimurium catalyzes
the transfer of the Genetic and biochemical data indicate that CheA is composed of at
least four structurally and functionally distinct
domains(1, 13, 14) . A phosphoaccepting
domain at the N terminus (H box-containing or H domain) is coupled via
a protease sensitive linker sequence to a CheY binding domain which is
in turn linked to an ATP binding/kinase domain (catalytic or C domain).
A fourth domain at the C terminus of the protein is required for
formation of ternary complexes with CheW and the receptor. The kinase
domain is homologous to corresponding domains in a large family of
histidine protein kinases that function in signal transduction to
provide phosphoryl groups for a second family of proteins with
regulator domains that are homologous to
CheY(15, 16) . Although the activity of each histidine
kinase is modulated by a different sensory input and each regulator
domain effects a different response, the chemistry of
autophosphorylation and phosphotransfer is conserved. In the case of
CheA (17, 18) as well as the osmosensory kinase,
EnvZ(19) , and the nitrogen regulatory kinase,
NRII(20) , it has been shown that autophosphorylation can occur
in trans with the kinase domain of one monomer catalyzing the
phosphorylation of a histidine residue in another monomer. Evidence has
been presented that NRII cannot catalyze the cis transfer of
phosphate from ATP to histidine by an intramolecular
route(20) . Here we report an investigation of the
phosphotransfer reactions catalyzed by purified CheA in the presence
and the absence of CheY. Using
The results were analyzed in terms of the reactions described in Table 2. It is assumed that rates of phosphotransfer are slow
compared with nucleotide binding and dissociation. Initial estimates
for dissociation constants for ATP and ADP were obtained by fitting
subsets of the data to the Michaelis-Menten equation by nonlinear
regression. Intercept replots were used to derive estimates of V
Figure 1:
The effect of CheA concentration on the
pseudo first order rate constant (k
where k
Figure 2:
The effect of CheA concentration on the
kinetics of CheA autophosphorylation at steady state. A coupled assay
(see text) was used to measure the rate of CheA autophosphorylation at
2 mM ATP and CheA from 0.08 to 5 µM (open
circles). The data were fit to a model that assumes that CheA is
in equilibrium between inactive monomer and active dimer with apparent K
Using
the ATPase assay we have found that CheA activity is severalfold higher
in the presence of potassium and ammonium cations compared with
reactions containing only sodium cations (NH
Figure 3:
Kinetics of CheA
Figure 4:
Effect of enzyme concentration on the
isotope exchange rate between ADP and ATP. The rate of exchange
catalyzed by CheA at concentrations ranging from 0.22 to 20 µM (at 211 µM [
Figure 5:
Isotope exchange between ATP and ADP
catalyzed by CheA. [
To
determine the values of the individual nucleotide binding constants and
the forward and reverse rate constants requires additional information.
This was obtained by measuring the V
Figure 6:
Steady state rate of CheA
autophosphorylation as a function of ATP concentration. A coupled assay
(see text) was used to measure the rate of CheA autophosphorylation
using ATP from 0.05 to 2.0 mM at 2 µM CheA in pH
7.5 reaction buffer (open circles) or 10 µM CheA
in pH 8.4 reaction buffer (closed circles). Plots of
[ATP]/v versus [ATP] were used to
determine K
Figure 7:
Dithiobis(succinimidyl propionate)
cross-linking of CheA dimers as a function of CheA concentration. The
reactions were carried out with 0.1-20 µM CheA, and
the extent of CheA dimer cross-linked was quantitated as described
under ``Materials and Methods.'' The amount of dimer was
normalized to the maximum amount of dimer observed under these
cross-linking conditions (
The CheA histidine kinase of E. coli and S.
typhimurium is a member of the large family of histidine protein
kinases involved in bacterial signal transduction. In cells CheA is
regulated in a complex with CheW and chemoreceptors so that its
activity reflects the signaling state of the receptor. To establish a
foundation for understanding the mechanism of CheA regulation, we have
characterized CheA activity using the purified protein. The kinetic
results obtained provide information about the basic mechanism of
histidine kinase autophosphorylation. Using phosphorous NMR we have
demonstrated that the site of CheA phosphorylation is at the N-3
position of histidine. This fits the hydrolysis data obtained for the
CheA phosphoramidate group under a variety of different conditions of
pH(4) . From hydrolysis data of the phosphohistidine in
homologous bacterial histidine
kinases(25, 26, 27) , it seems likely that
members of this family generally employ a mechanism involving
phosphorylation at N-3 of the histidine imidazole side chain. The
modified residue has been shown to co-chromatograph with
3-phosphohistidine in the case of the protein kinase PhoM(27) . Phosphohistidines generally have high phosphodonor potentials, and
it has been previously shown that histidine protein kinases can
catalyze an exchange of phosphoryl groups between ATP and ADP. Assuming
a ping-pong mechanism, the kinetics of the exchange reaction and the
kinetics of autophosphorylation can be used to estimate the binding
constants of ADP and ATP for both the phospho- and dephosphoenzymes.
The results indicate that ADP and ATP bind almost equally to
dephospho-CheA. This suggests that the Mg(II) coordination may involve
the We estimate that the turnover number of S. typhimurium CheA is approximately 10/min, and Tawa and
Stewart (28) have reported slightly lower values for the E.
coli enzyme. It seems likely that the phosphotransfer reactions
require the movement of the histidine group from a relatively
solvent-exposed position where it is free to bind to the active site of
a phosphoaccepting regulatory protein such as CheY to a position within
the active site of the kinase. The differences in ATP affinity for the
phospho- and dephosphoenzymes corresponds to a difference in standard
free energy of only about 1 kcal/mol, which argues strongly for the
notion that the phosphohistidine group has relatively little access to
the kinase active site where the nucleotide is bound. Exposure of the
phosphohistidine is also evidenced by the well documented observation
that response regulators can generally accept phosphoryl groups from
noncognate histidine kinases(16) . It has been shown that
response regulators can independently function to catalyze the transfer
of phosphoryl groups from phosphoramidate to the phosphoaccepting
aspartyl carboxylate(36) . Moreover, there is essentially no
affect of CheY binding to CheA on either the K CheA has previously
been shown to exist in a dimeric state at µM concentrations(21) . Here we show that this dimer
reversibly dissociates into an inactive monomeric form. The K The CheA monomer-dimer equilibrium appears
not to be affected by nucleotide binding, phosphorylation, or the
binding of CheY or CheW. One might expect that the interactions of the
phosphorylation site with the active site would contribute to the
stability of the dimer. This may not be the case, however. Findings
obtained from subunit exchange experiments with CheA Most
histidine kinases are receptors with a transmembrane topology similar
to that of the chemoreceptors that function to regulate CheA. Genetic
evidence suggests that at least in the case of EnvZ, receptor
dimerization is sufficient to cause kinase activation. NRII is a stable
dimer that like CheA is a cytosolic protein, but there is no evidence
that NRII kinase activity is regulated. Activation of CheA by
dimerization is insufficient to account for the activation of CheA when
it is in a complex with chemoreceptors and CheW, however. In fact, the
CheA-CheW-receptor ternary complex appears to be a stable 2:2:2 dimer,
and it has been shown that disulfide cross-linking of receptor subunits
within this structure does not preclude kinase regulation (41) .
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 939-945
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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 (CheA
2CheA; K
= 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 (K
values
0.2-0.3 mM), whereas ATP had a reduced affinity for
CheAP (K
0.8 mM)
compared with ADP (K 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 (k
10
min).
-phosphoryl group from ATP to one of its own
histidine residues,
His
(3, 4, 5) . CheA forms a
2:2:2 complex with an 18,000 molecular weight protein, CheW, and the
signaling domain of membrane chemoreceptors(6) . Within this
complex CheA autophosphorylation is regulated by the signaling state of
the receptor(7, 8) . Whether CheA is in a complex with
CheW and receptor or alone in solution, the phosphoryl group is rapidly
transferred from His
to an aspartate residue,
Asp
, in the chemotaxis response regulator protein,
CheY(4, 9, 10) . In its dephosphorylated
state CheY binds to CheA(11) . Phosphorylation of CheY reduces
its affinity for CheA (11) and increases its affinity for the
FliM protein at the flagellar basal structure(12) . There is
strong evidence to support the hypothesis that CheY
P binding to
FliM causes a tumbly motor response (reviewed in (2) ).
P NMR we show that the site
of phosphorylation on the histidine side chain is at the N-3 nitrogen
rather than the N-1 nitrogen as had previously been
reported(5) . At micromolar concentrations CheA has been shown
to be predominantly in a homodimeric form(21) . Here we show
that at submicromolar concentrations the monomer predominates (K
= 0.2-0.4
µM). A kinetic analysis of the rate of autophosphorylation
under these conditions indicates a second order dependence on CheA
concentration consistent with an intermolecular rather than an
intramolecular autophosphorylation mechanism. Adenine nucleotides and
CheY do not appear to affect the dissociation constant of the CheA
dimer. Our results are consistent with the notion that dimer formation
is an essential feature of CheA function in bacterial chemotaxis.
Protein Purification
All proteins were
derived from S. typhimurium genes expressed at high levels in E. coli as described previously. CheY(22) ,
CheW(23) , and CheA(15) . CheA
was purified
from E. coli DH5-
containing pME128 (pUC13 with a PstI-EcoRI CheA gene fragment that lacks the
first translational start site in CheA) by the same protocol as for
CheA. Protein concentrations were determined by the BCA assay (Pierce)
using bovine serum albumin as a standard. Values are expressed as
monomer concentrations.
Autophosphorylation of CheA was carried out by
dialyzing 20-30 mg of CheA against 4 liters of buffer containing
MgATP (50 mM Tris-HCl, pH 8.5, 20 mM KCl, 5.0
mM MgClP NMR of
CheA
P
, 0.10 mM 1,4-dithiothreithol, 1.0
mM ATP) overnight at 4 °C. The bulk ATP was removed from
the NMR sample by dialysis against the same buffer without ATP (3
4 liters). The sample was then dialyzed against buffer
containing EDTA (50 mM Tris-HCl, pH 8.5, 20 mM KCl,
0.10 mM 1,4-dithiothreithol, 10 mM EDTA). After
dialysis, the CheA samples were concentrated to 1 ml using an Amicon
Centricon with a molecular weight cutoff of 30,000. Deuterium oxide
(20%) served as a source of locked signal. The P NMR
spectra were obtained on a Bruker WM-250 at 101.2 MHz in 10-mm NMR
tubes. The spectra (16,000 data points) were typically acquired with
sweep widths of 5000 Hz at 4 °C. Chemical shifts were expressed
with respect to 85% phosphate as an external reference. After the
spectra were obtained, samples of the protein were analyzed by
SDS-polyacrylamide gel electrophoresis. No degradation of protein was
noted. To insure that any broadening of signals was not due to
paramagnetic impurities, buffers were treated with Chelex 1000.
MgCl
was recrystallized three times before being introduced
into the system. A synthetic peptide corresponding to the CheA
phosphorylation site
(NH-Arg-Ala-Ala-His-Ser-Ile-Lys-Gly-Gly-Ala-Gly-Thr-Cys-COOH)
was chemically phosphorylated by incubating 1.5 mM peptide
with 14.5 mM phosphoramidate in 50 mM Tris-HCl, pH
8.5, 0.20 mM 1,4-dithiothreithol, 10% DO
anaerobically under argon at room temperature. The reaction was
followed by obtaining spectra at 3-h intervals for 30 h. P
spectra for N-3-labeled histidine, phosphoramidate, and inorganic
phosphate were all observed (data not shown).
Kinetics of CheA
Autophosphorylation
Reactions were carried out at 23 °C
in pH 7.5 reaction buffer (50 mM Tris-Cl, pH 7.5, 100
mM potassium glutamate, 5.0 mM MgCl, 1.0
mM 1,4-dithiothreithol, 0.10 mM EDTA) containing 100
µM [-
P]ATP (Amersham Corp.;
specific activity, 1000-8000 cpm/pmol). Reactions were initiated
by the addition of ATP. Aliquots (10 µl) were added to 100 µl
of stop solution (100 mM EDTA, 0.2% SDS) and spotted onto
nitrocellulose using a Bio-Rad Bio-Dot slot blotter. The membranes were
washed with a solution of 20 mM Tris-HCl, pH 8, containing 150
mM NaCl and 10 mM sodium pyrophosphate, then washed
with ethanol, and dried. Spots were excised, and the levels of
radioactivity were assayed in a liquid scintillation spectrometer.
Kinetics of Phosphotransfer from CheA
CheAP to
ADP
P was prepared by incubating 34 µM CheA in pH 7.5 reaction buffer containing 0.80 mM [
-
P]ATP (Amersham Corp.; specific
activity, 7000 cpm/pmol) at 23 °C for 15 min, followed by dilution
with pH 7.5 reaction buffer without Mg
and
concentrating in Centricon-30 (Amicon). The dilution/concentration
procedure was repeated four times resulting in a final concentration of
[
-
P]ATP of <3 nM as measured by
radioactivity. Dephosphorylation reactions containing the indicated
concentrations of [
P]CheA
P in pH 7.5
reaction buffer at 23 °C were initiated by the addition of 100
µM ADP, and aliquots were removed at the indicated times
and added to stop solution. Initial levels of CheA
P were
determined by the addition of separate aliquots of
[
P]CheA
P and ADP to stop solution. The
reactions were blotted onto nitrocellulose and analyzed as described
for the autophosphorylation reactions.
CheA/CheY ATPase Kinetics
Rates of ATP
hydrolysis were measured in a spectroscopic assay for ATPase activity
as described previously(8) . The reaction mixtures contained
1.0 mM phosphoenolpyruvate, 0.20 mM NADH, 4 units of
pyruvate kinase, 4 units of lactate dehydrogenase (Boehringer
Mannheim), 25 µM CheY in pH 7.5 reaction buffer and the
indicated concentrations of ATP and CheA protein in a final volume of
0.10 ml. Reactions were conducted at 23 °C. NADH oxidation was
monitored at 340 nm in a Beckman DU-65 spectrophotometer. Reactions
were initiated by the addition of CheA and monitored for at least 5
min. The rate of ATP hydrolysis under steady state conditions was
calculated using 6220 M cm
for the extinction coefficient of NADH. In control experiments,
decreases of optical density to the zero value were generated in <2
s by the addition of 0.20 mM ADP, indicating that the coupling
reactions were not rate-limiting. The rate of ATP hydrolysis generally
attained a steady state within 1 min.
ATP/ADP Exchange Kinetics
Unlabeled ATP
and ADP (Sigma), [8-
C]ADP (ICN), and pH 8.4
reaction buffer (50 mMN-ethylmorpholine, pH 8.4, 60
mM KCl, 5.0 mM MgCl) were mixed at 23
°C, and CheA was added to initiate the exchange reaction. At the
indicated times, duplicate aliquots were quenched by the addition of an
equal volume of 1 N acetic acid containing unlabeled ATP and
ADP (to facilitate subsequent detection by UV; this addition had no
effect on the distribution of label). ATP and ADP were separated by
thin layer chromatography on polyethyleneimine-cellulose in 1 M acetic acid:4 M LiCl (4:1)(8) . Spots
corresponding to ATP and ADP were visualized by UV light (254 nm),
excised, and assayed for radioactivity in a liquid scintillation
spectrometer. At isotopic equilibrium, the label will be distributed
between ATP and ADP in proportion to their chemical concentrations and
from this the fraction of equilibrium attained (F) at any time (t) can be calculated(24) . Plots of ln(1 - F) versus time were linear, indicating that there was
no loss of enzyme activity during the course of the experiment.
Exchange rates (V) were calculated by the
equation:
(the maximal rate of exchange), K
k
/(k
+ k) , and Kk
/(k
+ k). Slope replots were used to obtain
estimates of K and K
.
These estimates were entered into the program MINSQ (MicroMath
Scientific Software, Salt Lake City, UT), which refines parameters
using nonlinear least squares analysis. The data were then fit to the
following relationship, which was simply derived from the reaction
scheme in Table 2:
Chemical Cross-linking of
CheA
Reactions were carried out at 23 °C in 25 mM Hepes-NaOH, pH 7.5, 100 mM KCl, 5.0 mM
MgCl with CheA at the indicated concentrations.
Dithiobis(succinimidyl propionate) (5.0 mg/ml in MeSO) was
added to a final concentration of 0.20 mg/ml. After 2 min the reactions
were stopped with the addition of Tris-HCl and lysine to final
concentrations of 50 mM and 10 mM, respectively.
Aliquots containing 185 ng of CheA from each reaction were analyzed on
8% SDS-polyacrylamide gel electrophoresis followed by transfer to
nitrocellulose (0.45 µm, Micron Separations Inc, Westboro, MA).
Protein was detected by Western blotting using rabbit polysera against
CheA. I-labeled goat anti-rabbit antisera (DuPont NEN)
was used as secondary antibody, and the blots were quantitated using a
Molecular Dynamics PhosphorImager.
CheA Autophosphorylation Occurs at the N-3 Position
of Histidine
In the initial report showing that the
phosphorylated residue in CheA is His, it was concluded
that the modification occurred at the N-1 position(5) . The
chromatographic procedures used in this analysis would not, however,
have been expected to resolve the N-1 and N-3 isomers. The rate of
hydrolysis of CheA
P as a function of pH is consistent with the
modification being at the N-3 rather than the N-1 position(4) ,
and the rates of hydrolysis of phosphohistidines in homologous
bacterial histidine kinases (25, 26, 27) have
given similar results. Moreover, using more refined chromatographic
procedures it was demonstrated that the phosphohistidine in the related
kinase PhoM is the N-3 isomer(27) . In order to conclusively
determine the site of phosphorylation in CheA, the
P NMR
spectrum of CheA
P was determined. The results (Table 1)
indicate that the chemical shift of the phosphoryl group in CheA
P
corresponds to that of an N-3 phosphohistidinyl residue rather than an
N-1 phosphoryl group. In addition, the same results were obtained for a
synthetic peptide corresponding to the phophorylation site in CheA that
was chemically phosphorylated using phosphoramidate. The NMR data
indicate that there is no other type of phosphorylated residue in
CheA
P.
Rates of CheA Autophosphorylation as a Function of
CheA Concentration
The rate of CheA autophosphorylation was
determined at different concentrations of CheA in the presence of 100
µM ATP (Fig. 1). Reactions were carried out at pH
7.5 under conditions similar to those used by Hess et
al.(9) . The effect of CheA concentration on the first
order rate constant for the reaction (k) was
determined from the slope of a semilogarithmic plot of the time course
of the reaction versus time (-ln((1 -
CheA
P)/CheA
P
) versus time) (28) . The molecular activity of CheA varied with
concentration. The data fit a mechanism where CheA is in equilibrium
between an inactive monomer and an active dimer with two independent
active sites (
)such that:
) of the CheA
autophosphorylation reaction. k
was determined
for reactions containing CheA from 0.2 to 8 µM and 0.1
mM [
P]ATP as described in the text. The line through the data represents a model that assumes that
CheA is in equilibrium between inactive monomer and active dimer with
apparent K
of 0.36 µM (see
text for details). The inset shows a semilogarithmic plot of
the time course of the reaction. The error bars represent
standard errors of the mean.
is the pseudo first order rate
constant at a given concentration of CheA, k
is
the maximum rate constant at high concentrations of CheA (i.e. when all the enzyme is a dimer, CheA), CheA is the total concentration of CheA, and K
is
the apparent dissociation constant for the CheA dimer. This analysis
gives a value of K of approximately 0.4
µM.Kinetic Characterization of ATP Hydrolysis Catalyzed
by CheA and CheY
The CheA/CheY phosphotransfer system
constitutes a signal transducing ATPase. To analyze this reaction we
have used a spectrophotometric assay that couples ATP hydrolysis to
NADH oxidation in an ATP-regenerating system using phosphoenolpyruvate,
pyruvate kinase, and lactate dehydrogenase. At sufficiently high
concentrations of CheY, CheA is dephosphorylated as fast as it is
phosphorylated, and the rate-limiting step is CheA autophosphorylation.
Under these conditions the dependence of CheA-mediated ATPase activity
on CheA concentration at saturating ATP and CheY indicates a second
order dependence at low CheA concentrations with an apparent K for dimer dissociation of 0.2 µM (Fig. 2), which is close to the value we estimated from
directly assaying CheA autophosphorylation in the absence of CheY (Fig. 1). These results lend further support to the conclusion
that CheA exists in an equilibrium between an inactive monomer and an
active dimer. It has previously been shown that CheY binds with high
affinity to the dephosphorylated form of CheA(11) . Because the
ATPase assays were performed with saturating CheY, the fact that
essentially the same apparent K for CheA dimer
dissociation was obtained by this procedure as when autophosphorylation
rates were measured in the complete absence of CheY indicates that CheY
binds equally to both the monomeric and dimeric forms of CheA.
of 0.17 µM (see text for
details). The rate of CheA autophosphorylation was also determined in
the presence of 5 µM CheA
(closed
circles).
> K
> Na
). This effect is
observed at high or low concentrations of CheA. Thus, monovalent
cations appear to affect the autophosphorylation reaction directly
rather than acting to alter the CheA monomer-dimer equilibrium. CheA
autophosphorylation has a pH optimum near 8.4(9, 29) .
The spectrophotometric assays reported here were performed mostly at pH
7.5. Experiments conducted at higher pH also indicated that this pH
effect was on CheA autophosphorylation rather than CheA dimerization or
ATP binding. The ATPase activity at 1.0 µM CheA was also
unaffected by addition of up to 20 µM CheW. CheW has been
reported to form a weak complex with CheA(21) , but it does not
appear that this interaction has any effect on CheA activity
independent of its involvement in the formation of the ternary complex
with the receptors.
Effect of CheA Dimerization on Phosphotransfer from
CheA
The transfer of phosphoryl groups from
CheAP to ADP
P to ADP can be readily assayed by first phosphorylating CheA
with
-
P-labeled ATP, isolating the
P-labeled CheA
P, and then measuring the loss of label
upon addition of ADP. A kinetic analysis of this reaction performed by
Tawa and Stewart (28) indicated that two processes were
involved. Whereas the majority of the CheA
P rapidly transferred
its phosphoryl group to ADP, a distinct slow phase was noted that
appeared to be due to a small fraction of CheA
P that slowly
converted from an ADP-unreactive to an ADP-reactive form. From our
results we would predict that this slow form should correspond to
monomeric CheA
P, which must be converted to dimer before it can
transfer its phosphoryl group to ADP. To test this possibility we have
measured the time course of CheA
P dephosphorylation as a function
of CheA
P concentration (Fig. 3). As predicted, the fraction
of CheA
P in the slowly reactive form does in fact depend on
CheA
P concentration. A quantitative analysis of our results gives
an estimated K
for CheAP of 0.2
µM. Thus, CheA phosphorylation does not appear to
significantly affect the monomer-dimer equilibrium.
P dephosphorylation
by ADP. The time course for CheA
P dephosphorylation was determined
at two concentrations of CheA
P: 0.25 µM (open
circles) and 4 µM (open squares). The rate
of dephosphorylation of 0.25 µM CheA
P was also
determined in the presence of 3.75 µM CheA
(closed circles).
Effects of CheA
The E. coli and S. typhimurium cheA genes contain a secondary translational start site that produces
an N-terminally truncated variant of CheA termed CheAon CheA
Activity that
begins at Met and therefore lacks the domain of CheA that
contains the phosphoaccepting histidine,
His
(30, 31) . It has previously been
shown that the kinase domain of CheA
retains an ability to
catalyze the phosphorylation of CheA proteins with an intact
phosphoaccepting domain(17, 18) . Thus,
CheA must be competent to form dimers with CheA, and we
would predict that at submicromolar concentrations of CheA where a
significant fraction of the protein is monomeric, the addition of
micromolar concentrations of CheA would stimulate kinase
activity through the formation of CheA-CheA heterodimers.
This supposition was verified using the ATPase assay to measure kinase
activity. At low CheA concentrations, the addition of relatively high
concentrations of CheA caused an increase in
autophosphorylation activity, and no concentration dependence of CheA
was observed over a range of concentrations (0.1-5
µM) when CheA was present at 5 µM (Fig. 2). CheA would also be expected to have
an activating effect on CheAP dephosphorylation by forming
CheA
-CheAP heterodimers and catalyzing phosphotransfer
to ADP. When the effect of CheA
was examined it was found
to cause a dramatic decrease in the amount of the slow phase component (Fig. 3).Kinetic Characterization of Kinase-catalyzed Exchange
of Phosphoryl Groups from ATP to ADP
Previous results had
indicated that CheA can catalyze isotope exchange between ATP and ADP (8, 32) . These findings, along with the isolation of
the phosphorylated enzyme (3) are consistent with a ping-pong
mechanism (23) for the histidine kinase ATP/ADP exchange
reaction. Our results indicated that the exchange reaction occurred
over a broad pH range (6.5-9.5) with an optima at pH 8.4, and in
addition the reaction was stimulated by potassium. These observations
are similar to results obtained by measuring rates of CheA
autophosphorylation(9, 29) . All exchange experiments
reported here were performed at pH 8.4 in the presence of potassium.
Isotope exchange rates were measured at different concentrations of
enzyme. The specific activity observed for CheA exhibited a
concentration dependence (Fig. 4) that fits the pattern expected
from an inactive monomer/active dimer equilibrium with a K for dimer dissociation of 0.4 µM.
Steady state rates of isotope exchange between ATP and ADP were
measured at 10 µM CheA where the protein is essentially
all in its dimeric form (Fig. 5). For ping-pong reactions,
reciprocal plots of rate versus varied substrate (ATP) at
different fixed substrate (ADP) should be parallel. Dead-end inhibition
by the fixed substrate results in deviations of the slopes and
inhibition by the varied substrates results in an upward curvature as
the line approaches the y axis. CheA showed dead-end
inhibition by both ADP and ATP. Thus, the unproductive complexes of ADP
bound to dephosphorylated enzyme and ATP bound to phosphorylated enzyme
appear to significantly affect the kinetics. The data were fit to the
isotope exchange rate equation for a ping-pong mechanism (see ), and values were obtained for constants that are
functions of the binding constants of ATP and ADP to the phospho and
dephospho forms of CheA as well as the rate constants for
phosphotransfer from ATP to CheA (k) and from
CheAP to ADP (k
) (Table 2).
C]ADP and 535
µM ATP). The data are expressed as the observed rates of
exchange (µM [C]ATP formed/min)
divided by the enzyme concentration versus enzyme
concentration. The data were fit to a model that assumes that CheA is
in equilibrium between inactive monomer and active
dimer.
C]ADP and ATP were varied,
and exchange rates were determined at 25 °C. CheA was at 10
µM. The lines are drawn to fit the entire set of
data for the rate equation (see ``Materials and Methods'').
ADP concentrations are as follows: 
, 31 µM; 
,
61 µM; 
, 93 µM; , 123
µM;
, 185
µM.
and K
for CheA autophosphorylation using the ATPase
assay: K = 0.33 mM and V = 11.0 min in pH 7.5
reaction buffer and K
= 0.27 mM and V = 7.4 min in pH 8.4 reaction buffer (Fig. 6). The pH profile with
maximum activity at pH 8.4 was observed for both reaction buffers. The
higher V
at pH 7.5 observed here is due to the
different reaction buffer components. According to our kinetic scheme,
the V equals k(CheA), and the K
corresponds to K. These relationships allow the
determination of all the kinetic constants (Table 2). The results
indicate that k
and k are
approximately equal. Tawa and Stewart (28) came to a similar
conclusion by directly comparing these rates. The values for these rate
constants are relatively low (7 min
). This slow
phosphotransfer rate tends to argue in favor of a central assumption in
our kinetic anlaysis, namely that nucleotide binding equilibria are
attained much more rapidly than phosphotransfer catalysis. The apparent
dissociation constants for ADP, approximately 0.2 mM, were not
significantly affected by CheA phosphorylation and are similar to the
apparent dissociation constant of ATP for the dephosphorylated enzyme.
Phosphorylation of CheA decreases its affinity for ATP by almost a
factor of three, indicating an unfavorable interaction between the
-phosphoryl group of the nucleotide and the phosphohistidine
moiety.
for ATP (0.33 mM (pH 7.5) and
0.27 mM (pH 8.4)).
Chemical Cross-linking of the CheA Dimer Is
Concentration-dependent
Kinetic results where CheA activity
was measured using several independent methods were all consistent with
CheA being a dissociable dimer (K =
0.2-0.4 µM) that is inactive in its monomeric state.
We were able to directly measure the CheA monomer-dimer equilibrium by
examining the effect of CheA concentration on the formation of
cross-links between monomers (Fig. 7). CheA at different
concentrations was cross-linked using dithiobis(succinimidyl
propionate), and the products were analyzed by electrophoresis in
polyacrylamide gels under nonreducing conditions. Only two species were
observed on cross-linking over a range of CheA concentrations
(0.1-25 µM). The low molecular weight species
corresponds to monomer with and without internal cross-links. The
second species co-migrated with molecular mass standards of
210-240 kDa. This high molecular weight form corresponds to
cross-linked dimer because partial reversal of the cross-linking
reaction with reducing agents revealed no intermediate forms. The
amount of cross-linked dimer formed was dependent on the concentration
of CheA in the reaction with the data fitting a theoretical curve
generated assuming a K for dimer dissociation of
0.3 µM.
70% of the total CheA). The data
represent the averages of two independent experiments. The error
bars represent standard errors of the mean. The line represents a model that assumes that CheA is in equilibrium
between inactive monomer and active dimer with a K
of 0.3 µM (see
text).
-
phosphates of the nucleotide rather than the
- phosphates. In this position it is doubtful that the bound
metal would play a central role in the phosphotransfer reaction. This
seems to be a general feature of protein kinases that work through a
phosphohistidine intermediate. For example in nucleoside diphosphate
kinase the
- coordination has been directly demonstrated by
x-ray crystallographic studies(33, 34, 35) .
Furthermore, the Myxococcus xanthus nucleoside diphosphate
kinase efficiently autophosphorylates with ATP in the absence of
magnesium(33) . for
ATP or the turnover number for phosphotransfer. for dimer dissociation under physiological
conditions was found to be 0.2-0.4 µM. The
possibility of intersubunit phosphorylation between the kinase domain
of one CheA subunit and the phosphohistidine domain of a second subunit
has previously been demonstrated using heterodimers of CheA mutants (17, 18) . Our data indicate that in the isolated
enzyme, the intersubunit mechanism is preferred by CheA. It has
previously been shown that for NRII, a CheA homolog involved in
nitrogen regulation, histidine autophosphorylation also involves an
intersubunit rather than an intrasubunit phosphotransfer
mechanism(20) . This aspect of histidine kinase function may
therefore be a conserved feature for most members of the histidine
kinase superfamily. In many respects, enzyme I involved in
phosphoenolpyruvate:carbohydrate phosphotransferase systems is
analogous to the CheA and the histidine kinase superfamily. This enzyme
catalyzes the phosphorylation of a histidine at the N-3 position using
phosphoenolpyruvate as a phosphodonor rather than ATP. Like CheA, the
site of phosphorylation is in one domain, and the phosphodonor
binding/dimerization functions are in a second domain(37) .
Furthermore, like CheA, the enzyme is inactive as a monomer and active
as a dimer(38) ., which
lacks the site of histidine phosphorylation, indicate that this
truncated protein is completely competent to function in the
phosphorylation of a variant with a defective kinase
domain(18) . Moreover, deletion of the domain of CheA that
couples the kinase to the chemoreceptors (with CheW) had no effect on
kinase autophosphorylation(13) . The finding that dimerization
is unaffected by ATP binding or by phosphorylation or even deletion of
the phosphoaccepting histidine supports the notion that the
phosphoaccepting histidine has very little contact with the kinase
active site. Clearly more work has to be carried out to establish the
nature of the interactions of the phosphorylation site with the active
site and how these interactions contribute to dimer stability.
)
We thank Pam Lane and Tanya Tolstykh for help with
preparation of proteins and members of the Stock lab for critical
reading of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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