(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 -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
CheA
P (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
).
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 -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) ).
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 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.
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 (the maximal rate of exchange), K
k
/(k
+ k
) , and K
k
/(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:
Figure 1:
The effect of CheA concentration on the
pseudo first order rate constant (k) 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.
where k 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.
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 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).
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 > 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.
Figure 3:
Kinetics of CheAP 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).
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 [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.
Figure 5:
Isotope exchange between ATP and ADP
catalyzed by CheA. [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.
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 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.
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 for ATP (0.33 mM (pH 7.5) and
0.27 mM (pH 8.4)).
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 (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).
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 -
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) .
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 for
ATP or the turnover number for phosphotransfer.
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 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) .
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, 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.
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) .