The ArsA ATPase is the catalytic subunit of a
novel arsenite pump, with two nucleotide-binding consensus sequences in
the N- and C-terminal halves of the protein. The single
tryptophan-containing Trp159 ArsA was used to
elucidate the elementary steps of the ATPase mechanism by fluorescence
stopped-flow experiments. The binding and hydrolysis of MgATP is a
multistep process with a minimal kinetic mechanism (Mechanism
1).
 |
INTRODUCTION |
Resistance to arsenical and antimonial salts in Escherichia
coli is conferred by the ars operon of conjugative
R-factor R773 (1). This operon encodes an ATP-coupled efflux pump that
actively transports the trivalent arsenicals and antimonials out of the cell; reducing the intracellular concentration of those metalloid oxyanions to subtoxic levels produces resistance (2). The pump is
composed of two subunits, ArsA, a 63-kDa catalytic subunit, and ArsB, a
45-kDa integral membrane protein that is the membrane anchor for
ArsA and the oxyanion-translocating sector of the pump (3). ArsA
can be purified as a soluble ATPase in the absence of ArsB (4). ArsA is
arranged into two homologous halves, the N-terminal (A1) (residues
1-282) and C-terminal (A2) (residues 321-583) domains, which are
connected by a flexible linker (residues 283-320) (5, 6). Each domain
contains the consensus sequence for the phosphate binding loop (P-loop)
of an ATP-binding site (7). Site-directed mutagenesis of these
sequences indicates that both of the putative nucleotide binding sites
are required for catalysis and resistance (8, 9). Intergenic
complementation and intragenic suppression studies on ArsA were
suggestive of a model in which a single catalytic site was formed at
the interface of an A1 and an A2 ATP binding site, possibly within a
homodimer (10, 11).
To investigate the interaction of nucleotides with the A1 and A2 sites,
we have conducted stopped-flow fluorescence experiments on ArsA. We
have previously shown that intrinsic tryptophan fluorescence can be
used to investigate the interaction of ArsA with its ligands (12).
However, the presence of multiple tryptophan residues in ArsA decreased
the signal-to-noise response to ligand binding, leading us to construct
single tryptophan derivatives of ArsA that are optically responsive to
the binding of substrates or products (13). Mutant arsA
genes were constructed containing single tryptophan codons (13).
Tryptophan residues 253, 522, and 524 were changed to tyrosine
residues, and a six-histidine tag was added to the C terminus,
producing a histidine-tagged ArsA containing only Trp159
(W159H6) (13). The single tryptophan-containing W159H6 ArsA gave
approximately a 4-fold increase in the signal-to-noise ratio in
response to MgATP/mol of tryptophan residue when compared with the wild
type enzyme (13). We have used this ArsA derivative for the transient
kinetic studies reported herein. It has proved possible to monitor the
interaction of MgATP with W159H6 by stopped-flow fluorescence
spectroscopy. Based on these analyses, we propose a model for the
mechanism of MgATP binding and hydrolysis.
 |
MATERIALS AND METHODS |
Purification of His6-tagged ArsA ATPase--
ArsA W159H6 ArsA
was purified as described previously (13), quickly frozen, and stored
in small aliquots at
80 °C. The concentration of purified ArsA was
determined by UV absorbance at 280 nm. The extinction coefficient for
W159H6 ArsA was calculated to be 20,250 M
1
cm
1 (14).
ATPase Assays--
A continuous assay was used to monitor
phosphate production by ArsA. Essentially, the absorbance change at 360 nm associated with the phosphorolysis of
2-amino-6-mercapto-7-methylpurine by the inorganic phosphate generated
by the ATPase activity was monitored (15). The phosphorolysis reaction
was catalyzed by purine nucleotide phosphorylase. The components of the
assay were provided as part of an EnzCheck phosphate assay kit
(Molecular Probes, Eugene, OR) and used according to the
manufacturer's recommendations. Assays were performed in 40 mM Tris-HCl (pH 7.5), 2 mM MgCl2, containing 0.2 mM sodium azide. The change in absorbance
with time was measured in a Unicam (UV2) UV-visible spectrometer.
Absorbance changes were converted into phosphate concentrations with
E360 = 12 mM
1
cm
1.
Fluorescence Measurements--
Fluorescence measurements were
performed in a Jasco FP777 spectrofluorometer maintained at 20 °C.
The excitation wavelength was set at 292.5 nm for selective excitation
of tryptophan fluorescence, and the emission was monitored at a
wavelength of 340 nm. The bandwidths for both emission and excitation
monochromators were 3 nm. The concentration of ArsA was 5 µM unless otherwise noted in 50 mM
MOPS1-KOH (pH 7.5), 0.25 mM EDTA.
In addition, time-resolved fluorescence measurements were also carried
out in an Applied Photophysics (London, UK) SX.18MV stopped-flow
instrument, operated at 20 °C. For measurements of the change in
tryptophan fluorescence, the samples were excited with light at 292.5 nm, selected with a monochromator, and the emission monitored at
wavelengths above 335 nm, using a cut-off filter. Invariably, equal
volumes of the reactants were mixed together in the stopped-flow
instrument, using two syringes of equal volume. Standard conditions for
multiple turnover experiments were 5 µM ArsA and for
single turnover experiments, 25 µM ArsA. All
concentrations are for the mixing chamber, unless stated otherwise, so
that the concentrations in the syringe were twice those quoted for the
mixing chamber.
Data Analysis--
The phosphate burst data was analyzed in
terms of an exponential plus linear function using the nonlinear
regression routine of the software package, SIGMAPLOT 4.0 (SPSS
Software Inc., Chicago, IL). Stopped-flow traces were analyzed by
fitting to single or multiple exponential functions, as appropriate,
using the nonlinear regression software with the Applied Photophysics
stopped-flow. Concentration dependence data were analyzed by nonlinear
regression fitting to hyperbolic functions, using SIGMAPLOT 4.0. Kinetic simulations were set up using the program Pro-K (Applied
Photophysics), which uses the Marquardt-Levenberg algorithm for global
optimization of the reaction parameters.
 |
RESULTS |
The Kinetics of ATP Binding and Turnover--
Previously we
established that the tryptophan fluorescence of ArsA (W159H6) was
sensitive to the binding of MgATP (13). Fig.
1A shows the change in
tryptophan fluorescence when 5 µM ArsA was manually mixed
with 1 mM MgATP; the reaction was initiated by the addition
of 5 mM MgCl2 to 5 µM ArsA, 1.0 mM ATP. There was a rapid biphasic increase in the
tryptophan fluorescence, to reach a plateau, before the fluorescence
decayed back to the baseline level (as defined by the fluorescence of 5 µM ArsA, 1.0 mM ATP), suggesting that little
product MgADP remains bound to the ArsA. However, the addition of EDTA
at the end of the reaction, to chelate the Mg2+, caused a
further decrease in tryptophan fluorescence, suggesting that some
product MgADP remained bound at the end of the reaction (Fig.
1A). A potentially plausible explanation for the
fluorescence profile of the reaction is that the binding of MgATP to
the ArsA induces an enhancement in the tryptophan fluorescence, which
remains constant until the ATP has been depleted, at which point the
fluorescence decays as the products are released. To test this
hypothesis, the mixing experiment was repeated with higher
concentrations of ArsA and ATP. The period during which the enhanced
fluorescence remained constant should have been diminished for higher
ArsA and extended for higher ATP if this phase was determined by the time taken for the steady-state turnover of the ATP. As shown in Fig.
1, B and C, no significant effect was observed
upon this phase with either higher ATP or ArsA concentrations,
respectively. This suggested that the enhancement in tryptophan
fluorescence was due to a transient build up and decay of a reaction
intermediate. The addition of excess EDTA rapidly reduced the ArsA
fluorescence, presumably due to the dissociation of ADP in the absence
of Mg2+ (Fig. 1), thus suggesting that the intermediate was
an ArsA-Mg2+ nucleotide complex because MgCl2
alone did not produce a change in the fluorescence of the ArsA (data
not shown). The addition of EDTA to ArsA or ArsA/Mg2+ did
not produce any change in the fluorescence of the ArsA and only a small
decrease with ArsA/MgADP (data not shown). This behavior suggests that
the intermediate was
ArsA-MgADP·Pi.2

View larger version (5K):
[in this window]
[in a new window]
|
Fig. 1.
ATP-induced conformational changes in
ArsA. ArsA was mixed with MgATP, in a Jasco FP777 fluorimeter, and
the tryptophan fluorescence changes monitored (excitation = 292.5 nm and emission = 340 nm). The conditions were as follows:
A, 5 µM ArsA, 1 mM ATP with 5 mM MgCl2; B, 5 µM
ArsA, 4 mM ATP with 5 mM MgCl2; and
C, 20 µM ArsA, 1 mM ATP with 5 mM MgCl2.
|
|
We have investigated the kinetics of formation of this intermediate in
detail using stopped-flow fluorescence spectroscopy. A typical
stopped-flow trace for the mixing of 5 µM ArsA, 0.5 mM ATP with 5 mM MgCl2 (mixing
chamber concentrations) is shown in trace A of Fig.
2. The trace was similar to that observed
in manual mixing experiments but the rapid-mixing experiment identified more complex behavior at shorter times. Over the first 10 s, the profile was clearly multiphasic with a very fast increase in
fluorescence, followed by a moderately fast decrease and then a slow
increase in fluorescence (Fig. 3).
Although the two fast phases merged, they were well resolved from the
slow phase. Accordingly, the initial part of the trace was analyzed as
a double exponential (during the first 4 s) and then as a single
exponential over the remainder of the trace. The three phases occurred
with rate constants of 52 s
1 (phase 1), 5.2 s
1 (phase 2), and 0.026 s
1 (phase 3),
respectively (Fig. 3). The entire data set was also fitted to a triple
exponential, which indicated similar rate constants of 48 s
1, 5.2 s
1, and 0.027 s
1 for
the three phases, respectively. The slow decay of the enhanced fluorescence back toward the baseline occurred with a rate constant of
approximately 2.4 × 10
3 s
1 (phase 4).
Previous studies apparently indicated that the slow decay in
fluorescence (phase 4) correlates with the rate of Pi production under limited turnover conditions (e.g. 5 µM ArsA and 20 µM MgATP), suggesting that
this phase is attributable to the product release step (13).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
ATP-induced conformational changes in
ArsA. Stopped-flow records for the mixing of 5 µM
ArsA, 500 µM ATP with 5 mM MgCl2
(A) and 5 µM ArsA with 500 µM
ATP, 5 mM MgCl2 (B). Changes in the
ArsA fluorescence were recorded with excitation = 292.5 nm and
emission > 335 nm.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
A stopped-flow record for the mixing of ArsA,
500 µM ATP with 5 mM
MgCl2 shown over four time bases that differ from one
another by an order of magnitude. The traces illustrate phase 3 (A), phase 2 and 3 (B), phase 1 and 2 (C), and phase 1 (D). The smooth curve through
each trace is the best-fit to a triple exponential equation with rate
constants of 48.0 (±1.02) s 1, 5.2 (±0.26)
s 1 and 0.027 (±0.0023) s 1. One vertical
division in traces A, B, C, and D represent a fluorescence change of
0.25, 0.5, 1.25, and 1.25%, respectively.
|
|
To clarify the mechanism, the dependence of the rate and amplitude of
each phase upon the ATP concentration was determined. The amplitude of
phase 2 decreased with the ATP concentration to a level around 200 µM ATP where it could no longer be distinguished from
phases 1 and 3. At these concentrations, the rate constant for phase 1 was determined either by fitting to a single exponential function over
the first few seconds of the trace or to a double exponential function
over the entire trace (e.g. 200 s). The rate constant
for the very fast phase (phase 1) increased in an apparently hyperbolic
manner with the ATP concentration up to a maximum around 1.5 mM and then decreased at higher concentrations (Fig.
4).3
The data obtained at ATP concentrations up to 1.5 mM could
be fitted to a hyperbola, indicating maximal and minimal rates of binding of 53.9 s
1 and 7.3 s
1, and a
Kd of 178 µM. A minimal value can be
calculated for the second-order association rate constant for the
binding of ATP to the ArsA from (kmax + kmin)/Kd, yielding a value of
0.34 × 106 M
1
s
1. For a fixed ATP concentration, the rate constant for
phase 1 was independent of the Mg2+ concentration (data not
shown). Over the ATP concentration range that phase 2 could be reliably
measured, the rate constant for this phase had little concentration
dependence, varying between 4.5 s
1 and 6.5 s
1 (Fig. 4,
). This behavior is consistent with the
simple three-step mechanism shown in Scheme
1, where the first step is a rapid
equilibrium with binding constant K2
(K2 = k
2/k2) followed by
transitions to states with high fluorescence enhancement
(e.g. ArsA4-MgATP) and lower fluorescence
enhancement (e.g. ArsA5-MgATP). The fluorescence
transient fits two exponential terms with observed rate constants,
k(1) = k
3 + k3[MgATP]/([MgATP] + K2) and k(2) = k4 + k
4. The ratio of
the amplitudes of the two exponential
phases at high substrate concentration will provide a measure of the
ratio of enhancements for the two states, calculated as 2.6 (16).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
The rates of MgATP-induced conformational
changes of ArsA. A series of stopped-flow records were generated
by pre-equilibrating ArsA with the indicated ATP concentration and then
mixing with 5 mM MgCl2 in a stopped-flow
device. The rates of the fast increase ( , phase 1) and decrease
( , phase 2) in the fluorescence of ArsA induced by the binding of
MgATP are plotted as a function of the ATP concentration. The rate
constant for phase 1 ( ) increases in a hyperbolic manner, and the
curve through the data points is the best-fit to a hyperbolic equation,
with minimal and maximal rates of 7.3 (±3.10) s 1 and
53.7 (±3.25) s 1, respectively, and a
Kd of 178 (±45.7) µM. The rate
constant for phase 2 ( ) was independent of the ATP concentration,
varying nonsystematically between 4.5 s 1 and 6.5 s 1.
|
|
The concentration dependence of the amplitude of phase 1 indicated an
overall equilibrium constant of 760 µM (Fig.
5). A plausible explanation for this
behavior is that the ArsA protein exists in two interconverting
conformations (e.g. ArsA1 and ArsA2)
that differ in their affinities for
ATP4 (see Scheme
2). The overall Kd is
a function of both K1 and
K2: Kd = K2(1 + K1)). Accordingly,
K1 can be calculated, from the overall
Kd and K2, as 3.33, indicating that 77% of the ArsA is in the ArsA1
conformation before the binding of MgATP. Because the rate of phase 1 increased with the ATP concentration but was independent of the
Mg2+ concentration, this implies that we were monitoring
the binding of MgATP to the ArsA. A possibility is that the
ArsA1 state is stabilized by the nonproductive binding of
ATP in the absence of Mg2+, which must dissociate before
MgATP can bind.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
An ATP titration curve for ArsA. A
series of stopped-flow records were generated by pre-equilibrating ArsA
with the indicated ATP concentration and then mixing with 5 mM MgCl2 in a stopped-flow device. The
amplitude of the stopped-flow signal for the fast increase in ArsA
fluorescence (phase 1), which is attributed to the binding of ATP to
the ArsA, is plotted as a function of the ATP concentration. The curve
through the data points is the best-fit to a hyperbolic equation with a
Kd of 760 (±45.7) µM.
|
|
Neither the rate constant for, nor the amplitude of, the slow increase
in fluorescence (e.g. phase 3) had any apparent dependence upon the ATP concentration (data not shown). The rate constant varied
nonsystematically between 0.025 and 0.05 s
1 (data not
shown). The rate constant for the slow decay in fluorescence back to
the baseline (phase 4) occurred with a rate constant of 2.3 × 10
3 s
1 that was also independent of the ATP
concentration (data not shown). Consequently suggesting that these
phases are attributable to a further isomerization of the
ArsA-nucleotide complex (e.g. the formation and decay of
ArsA6).
In a parallel set of experiments, the binding of MgATP to ArsA was
investigated. A typical stopped-flow trace for the mixing of 5 µM ArsA with 0.5 mM ATP,5 mM
MgCl2 is shown in Fig. 2B. The binding of MgATP
to ArsA was characterized by a slow, apparently monophasic,
fluorescence enhancement that occurred with a rate constant of 0.0334 s
1. Thus, this phase occurred at a rate comparable with
the slow fluorescence enhancement that occurred when the reaction was
initiated by mixing ArsA/ATP with Mg2+ (cf.
0.026 s
1). However, the fast increase (phase 1) and
decrease (phase 2) in fluorescence, which occurred within 4 s of
initiating the reaction with Mg2+, were not apparent. There
was an indication of a hyperbolic increase in the rate constant,
between 0.022 s
1 and 0.058 s
1, for the slow
enhancement in fluorescence when ArsA was mixed with MgATP. This
behavior is consistent with a two-step binding mechanism. The rapid
equilibrium binding of the ATP, which is rate limited by a slow
isomerization of the ArsA·ATP complex (e.g. k1,k
1
k2,k
2) is shown in
Scheme 3.
The data could be fitted to a hyperbolic equation for such a model:
kobs = k
2 + k2[MgATP]/([MgATP] + K1), yielding values for
K1, k2, and
k
2 of 624 µM, 0.038 s
1 and 0.022 s
1, respectively (Fig.
6A). The overall
Kd would be given by the following function:
Kd(overall) = K1/(1 + k2/k
2), indicating a
Kd of 232 (±75.5) µM. The amplitude
of the ATP-induced fluorescence enhancement increased in a hyperbolic manner with the ATP concentration, indicating an overall dissociation constant (Kd) of 427 (±62.3) µM (Fig.
6B). Hence, there is a small discrepancy between the
predicted and measured overall Kd, which could
readily be reconciled by postulating a further conformational
transition of the ArsA·MgATP complex. However, this seems unwarranted
at this time considering the difficulty in measuring the small increase
in the rate constants k2 and
k
2 and the consequent error in
K1. This behavior contrasts with that for the
Mg2+ initiated reaction, where the amplitude of the slow
increase in fluorescence was apparently independent of the ATP
concentration. A plausible explanation is that the MgATP initially
binds to an ArsA state that does not produce any change in fluorescence
but which is followed by a slow transition to a state with an enhanced fluorescence. When the ArsA was pre-equilibrated with Mg2+
before initiating the reaction by mixing with ATP, the traces were
similar to those generated by mixing ArsA with MgATP (data not shown).
This indicates that if Mg2+ can bind to ArsA in the absence
of nucleotides then this must be a rapid equilibrium process. These
differences in the kinetics of MgATP binding indicate that ATP can
bind to ArsA in the absence of Mg2+ to induce the
protein to adopt a different conformation.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
The concentration dependence of the binding
of MgATP to ArsA. 5 µM ArsA was mixed with the
indicated concentrations of ATP, 5 mM MgCl2 in
a stopped-flow device. The rate and amplitude of the resulting increase
in ArsA fluorescence are plotted as a function of the ATP
concentration. The curves through the rate and amplitude data sets are
the best-fits to hyperbolic equations with respective
Kd values of 623.7 (±179.0) µM and
427.3 (±62.3) µM.
|
|
The Binding of ATP under Single Turnover Conditions--
As shown
in Fig. 7A, when 25 µM ArsA (a 50 µM nucleotide binding site
concentration) was mixed with 25 µM MgATP the
stopped-flow trace generated was similar to those obtained under
multiple turnover conditions (e.g. 5 µM ArsA
with 500 µM MgATP). There was an increase in
fluorescence, which occurred with a rate constant of 3.6 × 10
2 s
1, and a subsequent decay in
fluorescence that occurred with a rate constant of 7.8 × 10
4 s
1. The protein fluorescence decayed
back to a level lower than the start of the trace, indicating that
there is a very fast increase in fluorescence probably due to the
binding of ATP. Indeed, we consistently noted a rapid increase in
fluorescence at the start of the single turnover traces that occurred
with a rate constant of approximately 20 s
1. In
comparison with multiple turnovers, the amplitude of the decay phase
was greater in relation to that of the enhancement phase, suggesting
that ADP remained bound to the ArsA after multiple turnovers.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7.
A single turnover of ATP by ArsA.
Stopped-flow records that show the change in ArsA fluorescence under
single turnover conditions. 25 µM ArsA (a 50 µM nucleotide binding site concentration) was mixed with
25 µM ATP, 5 mM MgCl2
(A), and 25 µM ArsA/25 µM ATP
was mixed with 5 mM MgCl2 (B). The
smooth curve through each record is the best-fit to a
double-exponential function with rate constants of 3.6 × 10 2 (±9.5 × 10 4) s 1
and 7.8 × 10 4 (±5.0 × 10 5)
s 1 (A), and a triple exponential function with rate
constants of 11.0 (±0.95) s 1, 7.6 (±0.90)
s 1, and 2.1 × 10 3 (±1.2 × 10 4) s 1 (B), respectively. One
vertical division represents an 0.25% change in the fluorescence of
ArsA.
|
|
In contrast, when 25 µM ArsA was incubated with 25 µM ATP before mixing with 5 mM
MgCl2 in the stopped-flow instrument, there was a rapid
increase in the protein fluorescence (phases 1 and 2), followed by a
slow decay (phase 4) (Fig. 7B). There was no slow increase
in fluorescence (phase 3). It was possible to fit the data to a triple
exponential function with the increase and the biphasic
decrease in fluorescence occurring with rate constants of 12.3 s
1 (phase 1), 6.22 s
1 (phase 2), and
2.0 × 10
3 s
1 (phase 4), respectively.
The rate constants for phases 1, 2, and 4 were comparable with the
calculated rate of ATP binding (cf. 14 s
1 for
25 µM ATP) and with the rate of fluorescence decay under multiple turnover conditions (cf. 4.5-6.5 s
1
for phase 2 and 2.3 × 10
3 s
1 for
phase 4), respectively. Although there was no slow enhancement in
fluorescence (e.g. phase 3) under these single turnover
conditions, the amplitude of the decay phases (e.g. phase 4)
when ArsA/ATP was mixed with Mg2+ was similar to that when
ArsA was mixed with MgATP (e.g. 3.0% versus
2.3% fluorescence change, respectively). This slow decay in
fluorescence cannot be attributed to multiple turnovers, which depleted
the concentration of this intermediate, during the latter stages of the
reaction, because this phase was independent of the ATP concentration
(cf. from measurements of single and multiple turnovers).
The slow increase and decrease in fluorescence presumably represents
the transient formation of an intermediate that forms and decays at
rates of 6.5 s
1 and 2.3 × 10
3
s
1, respectively. If this is an in-line intermediate then
the pre-equilibration with ATP (in the absence of Mg2+)
must induce the ArsA to adopt a similar conformation.
ADP Binding--
The binding of MgADP to ArsA was characterized
by a small enhancement in the tryptophan fluorescence, precluding a
rigorous analysis of the kinetics of binding. However, when ArsA was
mixed with a relatively high MgADP concentration (e.g. 10 mM), the stopped-flow traces generated were similar to
those for the binding of MgATP to ArsA, which had been pre-equilibrated
with ATP. There was a rapid increase and decrease in the fluorescence,
followed by a slow increase but not a subsequent decrease (Fig.
8). Recently, we have established that
the W141H6 ArsA mutant has greater optical sensitivity to the binding
of ADP, and a detailed analysis of the ADP binding mechanism for this
mutant will be presented elsewhere.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
The binding of ADP to ArsA. A
representative stopped-flow trace for the mixing of 5 µM
ArsA, 10 mM MgCl2 with 10 mM ADP,
10 mM MgCl2. The smooth curve through the data
is the best-fit line to a triple exponential function with rate
constants of 10.5 (±1.27) s 1, 9.28 (±1.31)
s 1, and 0.16 (±0.011) s 1, respectively.
One vertical division represents an 0.5% change in the fluorescence of
ArsA.
|
|
ADP Dissociation--
To test whether the dissociation of
MgADP was a rapid or slow process, the ArsA·MgADP complex was
chased with excess MgATP. Clearly, if MgADP dissociation was a rapid
process then we would have expected to observe an increase in the ArsA
fluorescence as the MgATP bound. On the other hand, if dissociation of
the MgADP was slow, then we would have expected to observe a decrease in the fluorescence as the MgADP dissociated. When 5 µM
ArsA was equilibrated with 50 µM ADP, 5 mM
MgCl2, and mixed with 500 µM ATP in a
stopped-flow device, there was a relatively slow decrease in
fluorescence, presumably as the ADP was displaced, followed by a slow
increase in fluorescence, presumably as the ATP was hydrolyzed (Fig.
9). The amplitude of the decrease in
fluorescence increased in a hyperbolic manner with the ArsA/MgADP
incubation time (Figs. 9 and 10),
indicating that the ADP induced a slow conformational change in the
ArsA, which occurred with a rate constant of 1.62 × 10
4 s
1. This behavior is consistent with
the increase in ArsA fluorescence that we have noted upon the binding
MgADP to ArsA. This conformational change was to a form that allowed
more rapid product release because the rate constant for the
dissociation process increased from 0.076 to 0.131 s
1
over the 8-h incubation time (Fig. 10).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 9.
ADP dissociation. 5 µM
ArsA was equilibrated with 50 µM ADP, 5 mM
MgCl2 for 1 min (A) or 120 min (B)
and then mixed with 500 µM ATP in a stopped-flow device
to displace the bound ADP.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 10.
The kinetics of a slow ADP-induced
conformation change in ArsA. 5 µM ArsA was
equilibrated with 50 µM ADP, 5 mM
MgCl2 for the indicated times before mixing with 500 µM ATP in a stopped-flow device to displace the bound
ADP. Both the rate and amplitude of the signal increased in an
exponential manner with respective rate constants of 1.8 × 10 2 (±5.0 × 10 3) s 1,
and 1.3 × 10 2 (±8.4 × 10 4)
s 1. The smooth curves through the data points are the
fits to a single exponential function.
|
|
Phosphate Burst--
A burst in phosphate production was
identified using a continuous assay to monitor phosphate release from
ArsA. As shown in Fig. 11, when 1 µM ArsA was mixed with 50 µM ATP there was
an exponential increase in the phosphate concentration during the first
400 s, followed by a linear steady-state release of
phosphate.5,6
The rate of the pre-steady-state phase only increased slightly with
increasing ATP concentration to a value of 3.7 × 10
3 s
1 for 300 µM ATP. The
pre-steady-state phase could not be readily resolved from the
steady-state phase for higher ATP concentrations. In conclusion, during
the phosphate burst approximately 2 nmol7 of phosphate were
released at a rate faster than 3.7 × 10
3
s
1 (for near saturating ATP concentrations).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 11.
Phosphate burst phase of ArsA ATPase. 1 µM ArsA was manually mixed with 50 µM ATP,
and the release of phosphate was monitored spectrophotometrically as
the change in absorbance at 360 nm associated with the phosphorolysis
of 2-amino-6-mercapto-7-methylpurine by phosphate. The curve through
the data is the best-fit to a single exponential plus a linear term
with a nonzero intercept: y = a(1 exp(b × t)) + (c × t) + d, where y is the absorbance at
time (t); a is the amplitude of the burst in
phosphate release; c is the linear rate of change in the
absorbance; and d is the absorbance at the start of the
experiment (zero time). A fit to this equation yielded values for
a, b, c, and d of 2.94 × 10 2 (±1.50 × 10 4)
absorbance/units, 1.14 × 10 3 (±8.5 × 10 6) s 1, 2.1 × 10 5
(±4.6 × 10 8) absorbance/units/s 1,
and 1.6 × 10 2 absorbance units, respectively. This
correlates with a 2.45 nmol pre-steady-state burst of phosphate release
at a rate of 1.14 × 10 3 s 1 and a
steady-state release of phosphate at a rate of 1.75 × 10 3 nmol s 1 nmol 1 of
ArsA.
|
|
The steady-state rate, as determined from the linear part of the
phosphate release time course, increased in a hyperbolic manner with
the ATP concentration to a maximal level around 200 µM
ATP and thereafter was subject to substrate inhibition (Fig. 12). Hence, values for
Vmax, Km, and
Ki, the substrate inhibition constant, were
determined from a fit of the data to the following equation for
substrate inhibition: v = Vmax[ATP]/Km + [ATP] + [ATP]2/Ki. This analysis yielded
values for Vmax, Km, and
Ki of 5.4 × 10
3 nmol
s
1·nmol
1 ArsA, 72 µM and
792 µM, respectively, and indicated a
kcat of 5.4 × 10
3
s
1 (single site catalysis) or 2.7 × 10
3 s
1 (two-site catalysis).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 12.
The steady-state kinetics of the ArsA
ATPase. The steady-state rate of ATP hydrolysis by ArsA was
determined from the linear rate of release of phosphate catalyzed by 1 µM ArsA. During the course of each measurement less than
10% of the ATP underwent hydrolysis, indicating that the measurements
were of true initial rates. The data was fitted to an equation for
substrate inhibition, yielding values for Vmax,
Km, and Ki of 5.4 × 10 3 (±8.8 × 10 4) nmol
s 1, 72 (±24.4) µM, and 792 (±297.7)
µM, respectively.
|
|
 |
DISCUSSION |
ArsA is the catalytic subunit of the arsenite transporter and is
thought to couple the hydrolysis of ATP to the movement of arsenicals
and antimonials through the membrane-spanning ArsB protein.
Consequently, knowledge of the ArsA ATPase mechanism will provide
information of fundamental importance in understanding the energy
transduction processes common to many transporters that are driven by
ATP hydrolysis, such as those belonging to the ABC superfamily.
Utilizing a derivative of ArsA that contains only a single tryptophan
residue, Trp159, which is optically responsive to the
binding of ATP, the ATPase mechanism of ArsA was investigated.
The kinetics of the binding of MgATP to ArsA were indicative of a
multistep mechanism. When ArsA was pre-equilibrated with ATP before
mixing with Mg2+ to initiate the reaction, the binding of
MgATP to the ArsA could be monitored as an increase in ArsA
fluorescence. The rate constant for the binding step increased
hyperbolically, indicative of a two-step process for the sequential
formation of ArsA3·MgATP and ArsA4·MgATP.
The forward and reverse rate constants for the formation of these
intermediates were calculated as k2 > 0.34 × 106 M
1 s
1,
k
2 = 7.3 s
1,
k3 = 53.9 s
1 and
k
3 = 7.3 s
1 (Schemes 1, 2, and 4
nomenclature). Formation of the ArsA4·MgATP complex was
followed by a further isomerization to the less fluorescent
ArsA5·MgATP state, at a rate of 4.5-6.5
s
1. There then followed a further conformational change
in which an intermediate with a higher fluorescence was formed at a
rate of 0.025-0.05 s
1. In comparison, when ArsA was
mixed directly with MgATP only a slow monophasic increase in the
fluorescence was observed, which occurred at a similar rate to the
formation of this intermediate. However, both the rate constant for,
and the concentration of, this intermediate increased in a hyperbolic
manner with the ATP concentration. This behavior is consistent with a
two-step binding process: the rapid equilibrium binding of ATP, to
produce an ArsA·MgATP state with unaltered fluorescence, followed by
a slow isomerization to a more fluorescent state. Accordingly, there
are several lines of evidence to indicate that ArsA adopts a different
conformation after equilibration with ATP. The binding of MgATP
directly to ArsA (
ATP state) is optically silent, whereas the binding
of MgATP to ArsA pre-equilibrated with ATP (+ATP state) induces an enhancement in fluorescence. It is conceivable that in the absence of
ATP the ArsA adopts a similar conformation to
ArsA5·MgATP, a "low-fluorescence" state. The
ATP
state has a lower affinity for MgATP (Kd = 624 µM) than the +ATP state (Kd = 178 µM). Pre-equilibration of the ArsA with ATP induces a
conformation that has low or no affinity for MgATP, the
ArsA1 state. The formation of this state is not apparent
from the kinetics of MgATP binding to ArsA or of ATP binding to
ArsA/Mg2+, suggesting that this is an ArsA1-ATP
state. Indeed, formation of this state could be the mechanistic basis
for the observed substrate inhibition at high ATP concentrations.
Irrespective of the mixing order, the slow formation of an intermediate
with enhanced fluorescence is observed under multiple turnover
conditions. The kinetic data is indicative of an intermediate that
builds up and decays with rate constants of 3.8 × 10
2 s
1 and 1-3 × 10
3
s
1, respectively (e.g. phases 3 and 4). This
cannot be the steady-state intermediate for the reaction; otherwise it
would only decay after depletion of the ATP. Accordingly, its formation
must precede the rate-limiting step of the reaction. This intermediate
has greater fluorescence than the ArsA·MgADP complex suggesting that it is the ArsA-MgADP·Pi complex. An alternative
possibility is that it is a state with a higher affinity for ADP
relative to that of the final state. Consistent with the former
interpretation, phosphate is released with a relatively slow rate
constant (e.g. koff = 3.7 × 10
3 s
1 for the phosphate burst with 300 µM ATP) compared with the rate of formation of this
intermediate (e.g. k = 3.8 × 10
2 s
1). The slow release of phosphate
suggests that there would not be an appreciable build up of
ArsA·MgADP during the time course of the build up of the intermediate
with enhanced fluorescence. We tentatively conclude that this
intermediate is the ArsA-MgADP·Pi complex. We have
previously measured phosphate production during a 4-fold limited
turnover of ATP by ArsA (13). A discontinuous assay was used to monitor
the hydrolysis of 20 µM ATP by 5 µM ArsA,
with the reaction terminated at set times with trichloroacetic acid to
displace bound Pi. We found that during the period of enhanced fluorescence (phase 3, 100-200 s), there was a stoichiometric production of bound phosphate, consistent with the proposal that phase
3 is attributable to the production of ArsA-ADP·Pi. The slow decay in fluorescence is presumably attributable to the product release steps. ADP is released at a much faster rate than the decay in
fluorescence (e.g. for ADP koff
0.08 s
1, whereas kobs for phase
4 = 1-3 × 10
3 s
1). On the other
hand, the decay in fluorescence occurs at a rate similar to that for
phosphate release (e.g. for Pi
koff
3.7 × 10
3
s
1, whereas kobs for phase 4 = 1-3 × 10
3 s
1). If this is the
case, then hydrolysis of the ATP must be fast because the phosphate
burst includes both the hydrolysis and phosphate release steps. An
alternative possibility is that the fluorescence decay is due to a
conformational change subsequent to the release of the phosphate.
However, the fact that there is a phosphate burst implies that
phosphate release is more rapid than the step that is rate-limiting in
the steady-state. Because neither the hydrolysis or product release
steps are rate-limiting for the steady-state, we conclude that there is
a rate-limiting conformational change in ArsA following product release.
The stoichiometry of the phosphate burst is 1.7 (±0.28), suggesting
that both nucleotide sites of ArsA are catalytic. Moreover, the
steady-state rate for a single site is apparently faster than the rate
constant for the phosphate burst and phase 4 of the fluorescence profile, whereas these steps in the reaction mechanism must be faster
than the rate-limiting step. However, if both sites were catalytic,
this would in effect halve kcat, which would
then have a value reasonably consistent with the other kinetic data.
Indeed, computer simulations revealed that the formation of the
ArsA-MgADP·Pi intermediate (with enhanced fluorescence)
need only be followed by the relatively fast formation of a further
intermediate (e.g. 10× the rate constant for phase 4) that
decays with a rate constant marginally slower than does
ArsA-MgADP·Pi. Under these conditions an intermediate
would build up and decay at the rates of phases 3 and 4. Thus, there is
kinetic evidence to support the supposition that both the A1 and A2
sites of ArsA bind and hydrolyze ATP. Previous studies have shown that
the A1 and A2 sites can be covalently labeled with ATP and FSBA,
respectively, indicating that both sites are available for nucleotide
binding (17, 18). However, it was proposed that antimonite might act as
a switch in regulating ATP binding to the A2 site (18). Our studies do
not support this supposition; we find no evidence for two sites of
differing affinity, and the data suggest that both sites are
catalytically competent.
It was possible to simulate the time for the fluorescence changes
associated with the binding of ATP to ArsA (e.g. 1 mM ATP to 5 µM ArsA) using the measured rate
constants and the minimal kinetic mechanism shown in Scheme
4 with the kinetic constants, k2 = 0.04 µM
1
s
1, k
2 = 7.3 s
1,
k3 = 54 s
1,
k4 = 6.5 s
1,
k5 = 0.04 s
1,
k
5 = 0.02 s
1,
k6 = 0.004 s
1,
k7 = 0.08 s
1,
k8 = 0.001 s
1,
k1 = fast.8 The model allows for
the formation of an intermediate (e.g.
ArsA6-MgADP·Pi) during the first 100 s
and its decay over the following 900 s and for a pre-steady state
burst and subsequent steady-state release of phosphate (Fig.
13). The fluorescence of the
intermediate decays to about 0.33-fold that of the maximum due to the
formation of the subsequent steady-state intermediate (e.g.
ArsA7), which only decays after all of the ATP has been
depleted. Under single turnover conditions, there is a decay in the
fluorescence of the ArsA6-MgADP·Pi
intermediate to near the baseline because there is no substantial build
up in the steady-state intermediate (Fig. 13). The model incorporates a
number of simplifications and assumptions; first, that hydrolysis
occurs at step 5, whereas equally this could occur at step 4, which
would then be followed by a slow isomerization between different
conformations of the ArsA-ADP·Pi complex. In any event,
the hydrolysis step is faster than the subsequent product-release steps
and the overall mechanism would be similar. The build up of the
ArsA6-MgADP·Pi intermediate requires the
subsequent formation (at moderate rate compared with
k5) and decay (at a slower rate than
k5) of a steady-state intermediate. To minimize
the number of mechanistic steps, we propose the ordered dissociation of
phosphate followed by ADP, leading to the build up of the
ArsA7 steady-state intermediate. Both phosphate and ADP
release precede the steady-state step, which we propose to be a
conformational change in the ArsA protein. The model predicts a return
to baseline fluorescence as the ATP is depleted at the end of the
reaction, because we have made ADP dissociation irreversible. However,
ArsA can bind MgADP, and this is a multistep process. We do not observe a decay in the fluorescence to the baseline under multiple turnover conditions probably because such behavior would be masked by the increase in fluorescence due to the binding of MgADP. As a further test
of the validity of the model, we attempted to fit a stopped-flow fluorescence trace directly to kinetic Scheme 4. Clearly, for a single
trace the problem would be too ill-conditioned to identify a unique
solution in which all the parameters (e.g. the rates of
interconversion of the different ArsA states and their relative fluorescence values) were allowed to simultaneously vary during the
nonlinear fitting procedure. Instead, the rates were held constant, and the relative fluorescence values of the different ArsA
states were optimized. The fitting procedure indicated relative fluorescence values for ArsA2, ArsA3-MgATP,
ArsA4-MgATP, ArsA5-MgATP,
ArsA6-MgADP·Pi, ArsA7-MgADP, and
ArsA7 of 1.00, 0.995, 1.052, 1.041, 1.037, 1.797, and
1.019, respectively. This analysis suggested that the maximal
fluorescence enhancement observed 100 s after mixing ArsA with
MgATP was attributable to the build up of
ArsA6-MgADP·Pi (e.g. 3 µM) and ArsA7-MgADP (e.g. 0.08 µM).9 Although
there is only a slight build up of ArsA7-MgADP, its high
fluorescence enhancement (e.g. 79.7%) is a significant contribution to the overall enhancement. Fig.
14 shows the best-fit curve
(e.g. curve A) superimposed upon a semi-logarithmic plot of
a stopped-flow trace. There is a deviation of the best-fit curve from
the measured data toward the end of the trace that is probably
attributable to the fact that the decay in fluorescence is biphasic
(rather than monophasic). To test this hypothesis, we introduced a
further step (e.g. an isomerization of ArsA2)
and allowed a free fit of the rate constants for these last two steps
(e.g. steps 8 and 9), indicating respective values of 1.3 × 10
2 (±2.3 × 10
3)
s
1 and 4.2 × 10
5 (±1.7 × 10
5) s
1. Clearly the latter step is too
slow relative to the steady-state rate to be an in-line intermediate
but could be an isomerization to a different catalytic form of ArsA,
such as ArsA1. We conclude that kinetic Scheme 4 provides a
minimal model to account for both the kinetics of the ArsA catalyzed
ATPase reaction and of the fluorescence profile that results from the
ATP-induced conformational changes in ArsA.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 13.
A simulation of the kinetic mechanism of
ArsA. To investigate whether the measured rate constants could be
assimilated into a coherent scheme that would predict the observed
appearance of a pre-steady-state intermediate, a computer simulation
was performed on kinetic Scheme 4 for the following reaction
conditions: 5 µM ArsA with 1 mM MgATP
(multiple turnover) (A); and 25 µM ArsA with
25 µM MgATP (single turnover) (B). The changes
in the concentration of the ArsA-ADP·Pi intermediate
(A) and phosphate (B) concentration with time are
shown. The vertical axis represents the change in concentration of
species in µM.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 14.
A simulation of the fluorescence profile for
the ATP-induced conformational changes in ArsA during catalysis. A
semi-logarithmic plot of a stopped-flow trace generated by mixing 5 µM ArsA with 1 mM MgATP is shown. The smooth
curves through the stopped-flow trace are the best-fits to the kinetic
model developed for the ArsA ATPase reaction (e.g. Scheme 4
under "Discussion"). For curve A the rate constants for
the kinetic scheme were fixed, and the fluorescence values for the
different ArsA states were optimized during the fitting procedure.
Curve B was generated using a model for which an extra
isomerization of ArsA7 was included in kinetic Scheme 4.
For curve B the rate constants for the final two
(isomerization) steps and the fluorescence values for the different
ArsA states were optimized during the fitting procedure. Visual
inspection of the curves indicates a better fit of curve (B)
to the data than curve (A) (which is accompanied by a
1.3-fold improvement in the residual variance). One vertical division
represents a 1.0% change in the fluorescence of ArsA. The reaction
time is presented in logarithmic progression on the horizontal
axis.
|
|