From the Division of Infection and Immunity,
the Institute of Biomedical and Life Sciences, Robertson Building,
The University of Glasgow,
Glasgow G11 6NU, Scotland, United Kingdom and the
¶ Department of Biochemistry and Molecular Biology, Wayne
State University School of Medicine, Detroit, Michigan 48201
Received for publication, September 5, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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ArsA is the catalytic subunit of the arsenical
pump, coupling ATP hydrolysis to the efflux of arsenicals through the
ArsB membrane protein. It is a paradigm for understanding the
structure-function of the nucleotide binding domains (NBD) of medically
important efflux pumps, such as P-glycoprotein, because it has two
sequence-related, interacting NBD, for which the structure is known. On
the basis of a rigorous analysis of the pre-steady-state kinetics of
nucleotide binding and hydrolysis, we propose a model in which ArsA
alternates between two mutually exclusive conformations as follows: the
ArsA1 conformation in which the A1 site is closed but
the A2 site open; and the ArsA2 conformation, in which the
A1 and A2 sites are open and closed, respectively. Antimonite elicits
its effects by sequestering ArsA in the ArsA1 conformation,
which catalyzes rapid ATP hydrolysis at the A2 site to drive ArsA
between conformations that have high (nucleotide-bound ArsA) and low
affinity (nucleotide-free ArsA) for Sb(III). ArsA potentially utilizes
this process to sequester Sb(III) from the medium and eject it into the
channel of ArsB.
One of the most frequently employed strategies to gain
resistance to cytotoxic compounds in both eukaryotes and prokaryotes is
the active extrusion of these compounds from the cell to reduce the
intracellular concentration to subtoxic levels (1). Protein pumps that
span the membrane catalyze this extrusion process, and many of them
belong to the ATP-binding cassette
(ABC)1 superfamily (2). This
family includes the human multidrug resistance P-glycoprotein, which
confers resistance to anti-cancer drugs (2), and homologues from
bacteria (3, 4), fungi (5), and protozoa (6). Generally, these ABC
transporters are composed of two homologous halves, each containing two
parts as follows: a transmembrane domain putatively arranged into 6 Our understanding of the mode of operation of these efflux pumps is
still rudimentary, and several fundamental questions concerning their
function need to be answered if we are to design inhibitors that block
these pumps, allowing us to overcome multidrug resistance. For example,
since most, if not all, ABC drug transporters have two
nucleotide-binding sites (NBS), it is of importance to determine whether these sites are functionally equivalent. Biochemical studies of
P-glycoprotein indicate that both NBS can hydrolyze ATP, but substrate-stimulated ATPase activity requires interaction between the
two halves of the molecule (7). Inactivation by chemical modification
or mutagenesis of either NBS causes a loss of activity, suggesting
cooperativity between the sites (8, 9), and it has been suggested that
the sites function by an alternating site mechanism (10).
P-glycoprotein has been overexpressed and can be purified as a
functional ATPase in low concentration of detergent, enabling
biophysical studies of the protein (11). The binding of fluorescent
analogues of ATP to the protein indicated that there is a single class
of site, suggesting that the two NBS are equivalent. Perhaps this is
not surprising because the NBS have similar sequences. Although the NBS
of P-glycoprotein resemble one another, this is not the case for other
mammalian ABC drug transporters, such as multidrug resistance protein
1, and recent studies have shown that the NBS of multidrug resistance
protein 1 are nonequivalent (12, 13). Clearly, to understand fully how
the NBS interact will require structural information. A recent advance
in our understanding has come from the determination, by x-ray
crystallography, of the structure of HisP (14, 15), the ATPase subunit
of the histidine permease, a bacterial ABC transporter that catalyzes
the uptake of histidine. Although arranging the monomeric HisP into a
dimer is speculative, the structure predicts that the two
nucleotide-binding sites are orientated away from one another.
Inactivation of one of these sites results in a transporter that has
half the ATPase and transport activity of the wild-type protein complex
(16), suggesting that there is little or no cooperativity between these
sites. This may represent a real difference with P-glycoprotein,
because cross-linking studies have shown that the NBS of P-glycoprotein
are in close proximity (17). In addition, the NBD of P-glycoprotein has
a site for ligands, such as flavonoids, that modulate drug transport,
probably by inhibiting the ATPase activity (18). Unfortunately, we do not have a view of the structure of P-glycoprotein at atomic
resolution, and at the present we must glean information from other
efflux pumps that have proved more amenable to structure-function studies.
The ArsAB ATPase is a prokaryotic pump that exhibits structural and
functional similarity to P-glycoprotein (19). They are both efflux
pumps for multiple forms of toxic compounds, have two similar consensus
nucleotide binding domains, are substrate-dependent ATPases, have 12 membrane-spanning Previously, we established that the tryptophan fluorescence of W159H6
ArsA was far more responsive to the binding of the substrate MgATP than
the product MgADP, allowing us to exploit this behavior in elucidating
the substrate binding, but not the product release, steps of the ATPase
mechanism by stopped-flow fluorescence spectroscopy (19, 26, 27). In
contrast, the W141H6 ArsA is more responsive to the binding of the
product MgADP than the substrate MgATP (28), and here we exploit this
behavior in elucidating the product release steps of the ATPase mechanism.
Purification of His6-tagged ArsA ATPase--
W141H6
ArsA and W159H6 ArsA were purified as described previously (28),
quickly frozen, and stored in small aliquots at Fluorescence Measurements--
An Applied Photophysics
(London, UK) SX.18MV stopped-flow instrument, operated at 20 °C, was
used to monitor ligand-induced changes in the steady-state fluorescence
of ArsA with time. 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. For measurements of the change in MANT
fluorescence, the samples were excited with light at 292.5 nm, selected
with a monochromator, and the emission monitored at wavelengths above 420 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. The concentration of ArsA was 5 µM, unless otherwise noted, in 50 mM MOPS-KOH
(pH 7.5), 0.25 mM EDTA. 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. The
base-line fluorescence was generally set by mixing 5 µM
ArsA with buffer and increasing the photomultiplier tube voltage to a
level that would give a 4-V signal and ligand-induced changes in
fluorescence measured relative to this signal (i.e. an
increase in the signal from 4 to 4.1 V would correspond to a 2.5%
increase in fluorescence and from 4 to 3.9 V a 2.5% quench in
fluorescence). Since some of the experiments were conducted over a long
time base, we decided to test for photobleaching of the protein by
monitoring the fluorescence of ArsA, mixed with buffer in the
stopped-flow instrument, over 1000 s; no decay in the protein
fluorescence was observed over this time. As a routine second check of
fluorescence signals that decay over 1000 s, we have closed the
lamp shutter during data acquisition for about 100-200 s and then
re-opened it before the end of the reaction. In this manner we have
been able to check that the signal continues to decay in the absence of
light, indicating that it represents a true ligand-induced change in
the protein fluorescence rather than photobleaching. Since ADP
inner-filter effects would tend to reduce the signal size for the high
concentrations of ADP used in some of the assays, we conducted the
following experiment to measure the inner-filter effect for a series of ADP concentrations, bovine serum albumin was mixed with increasing concentrations of ADP up to 5 mM, and the drop in base-line
signal was recorded. An inner-filter effect was observed, but it was much less severe than observed in a conventional fluorimeter, increasing linearly over the studied range, with concentrations below 5 mM decreasing the base-line signal by less than 10%. The experiment was also repeated with ArsA W159H6, which is optically unresponsive to the binding of ADP (28), indicating a similar inner-filter effect. Accordingly, data points were corrected for this
small inner-filter effect.
Data Analysis--
Stopped-flow traces, generated using a
logarithmic time base (29), were analyzed by fitting to single
(e.g. s = A·exp 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 (26). 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
MgADP Binding to ArsA--
The binding of MgADP to W141H6 ArsA
causes a quench in the tryptophan fluorescence of the protein that can
be time-resolved by stopped-flow fluorescence spectroscopy. Fig.
1 shows representative stopped-flow
records for the mixing of 5 µM W141H6 ArsA with ADP/5 mM MgCl2. In each case the profile was clearly
multiphasic, with a very fast (t1/2 <2 ms) phase
that was well resolved from a slow decay over 1000 s. Overall, the traces could be fitted to a 4-exponential function, indicative of four
phases with t1/2 <1 ms, t1/2 <2
s, t1/2 <40 s, and t1/2 <400 s,
respectively. However, we found that as the ADP concentration was
increased a 5-exponential function gave a better fit because of the
appearance of a phase with a t1/2 <3 ms. Although
this phase involved an increase in fluorescence, this was only slight
and not well resolved from the very fast decrease in fluorescence at
low ADP concentrations.
As shown in Fig. 1, only the rate and amplitude of the very fast
phase varied with the ADP concentration, indicating that the slower
phases were due to isomerizations of the ArsA-ADP complex. The rate
constant for the very fast phase increased in a hyperbolic manner with
the MgADP concentration (Fig.
2A). A fit of the rate data to
the following hyperbolic Equation 1,
Dissociation of the ArsA-MgADP Complex--
We attempted to
measure the rate constant for the dissociation of MgADP from the
ArsA-MgADP complex by displacement with MANT-ADP. As shown in Fig.
3A, when 5 µM
ArsA, 0.5 mM MgADP was mixed with 125 µM
MANT-ADP, 5 mM MgCl2 there was an increase in
MANT fluorescence, indicating that the ADP had been replaced. However,
an identical trace was obtained when the ADP was included with the
MANT-ADP (Fig. 3C), rather than with the ArsA, during the
mixing experiment. This behavior suggests that the ADP can rapidly
dissociate from the ArsA and competes with MANT-ADP for binding to
ArsA. For this to occur, ADP dissociation must be faster than the
binding of MANT-ATP to ArsA (e.g. 223 s Mg2+ Binding to ArsA-ADP--
In a parallel set of
experiments, the binding of Mg2+ to the W141H6 ArsA-ADP
complex was investigated, revealing a similar behavior; formation of
the ArsA-MgADP complex led to a fast quench in fluorescence, followed
by a slow decay over several minutes (Fig.
4). Only the rate of the fast phase was
dependent upon the ADP concentration, suggesting that the three slow
phases are attributable to isomerizations of the ArsA-MgADP complex
(data not shown). The rate of the fast phase increased in a hyperbolic
manner (Fig. 5A), deviating
only slightly at high concentrations, and the early data points were best fit to a hyperbolic equation with a zero intercept (Equation 4),
The relatively slow rate constant for formation of the ArsA-MgADP
complex from ArsA-ADP and Mg2+ (e.g.
kmax = 170 (± 10)
s ADP Dissociation from the ArsA-ADP Complex--
To test the
prediction that ADP dissociation from ArsA2 becomes
rate-limiting at high ADP concentrations, we attempted to measure the
ADP dissociation rate constant, in the absence of Mg2+, by
displacement with MANT-ADP. The binding of MANT-ADP to ArsA induced a
rapid increase in MANT fluorescence, followed by a slower increase,
which was best fit to a 4-exponential function, yielding a rate
constant of 350 (± 10) s
The data presented herein supports the existence of an ArsA-ADP
complex, and the value obtained for the rate constant for the
dissociation of ADP is comparable with that determined for the maximal
rate of formation of the ArsA-MgADP binary complex from ArsA-ADP and
Mg2+. Accordingly, this behavior is consistent with the
rate-limiting dissociation of ADP from ArsA during the formation of the
ArsA-MgADP complex from ArsA-ADP and Mg2+.
Mg2+ Dissociation from the ArsA-MgADP Complex--
The
decrease in the tryptophan fluorescence upon formation of the
ArsA-MgADP complex can be reversed by mixing with EDTA, suggesting that
ADP is released as EDTA chelates the Mg2+. The stopped-flow
trace shown in Fig. 7B, for
the mixing of 5 µM ArsA, 0.5 mM ADP, 5 mM MgCl2 with 20 mM EDTA, was best
fit to a double exponential function, with rate constants of 6.6 and 0.17 s Sb(III) Binding to ArsA--
The binding of Sb(III) to ArsA
induced a small quench in the tryptophan fluorescence, indicative of
the formation of an ArsA-Sb(III) complex (Fig.
8A). The stopped-flow traces
were best fit to a triple exponential function with rate constants of
about 20, 0.3, and 0.03 s Sb(III) Binding to the ArsA-MgADP Complex--
The binding
of Sb(III) to the ArsA-MgADP complex was characterized by a rapid
decrease in tryptophan fluorescence (e.g.
t1/2 <50 ms,
The kinetics of formation of the ArsA-MgADP-Sb(III) complex are similar
to those observed when Sb(III) was mixed with ArsA/MgATP (27). The
addition of MgATP to Trp159 ArsA caused a transient
increase in the tryptophan fluorescence, which we attributed to the
build up of the ArsA-MgADP·Pi complex. The addition of Sb(III) to
this complex rapidly reversed the fluorescence enhancement, suggesting
that Sb(III) caused destabilization of the complex, and the rate of
this process increases in a hyperbolic manner with the Sb(III)
concentration (i.e. kmax = 99 s Mg2+ Binding to the ArsA-ADP-Sb(III) Complex--
As
shown in Fig. 10, there was little
difference in the signal amplitudes for the binding of Mg2+
to the ArsA-ADP and ArsA-ADP-Sb(III) complexes, apart from a 3%
decrease in signal in increasing the Sb(III) concentration from 25 to
2500 µM. These traces indicate that the quench in
fluorescence produced by the binding of Sb(III) to ArsA-MgADP is not in
fact additive but tends to reduce slightly the overall signal that predominantly arises from the addition of Mg2+ to the
complex. The formation of the ArsA-MgADP-Sb(III) complex occurred at a
slower rate than that of the ArsA-MgADP complex, but there was no
apparent dependence upon the Sb(III) concentration, and the rate of the
fastest phase varied nonsystematically between 220 and 340 s Mg2+ Dissociation from the ArsA-MgADP-Sb(III)
Complex--
The effects of Sb(III) upon the rate of MgADP
dissociation were investigated by dissociating the ArsA-MgADP complex
with EDTA in the presence of Sb(III). The stopped-flow trace shown as
Fig. 7C indicated that Sb(III) slowed the rate of
dissociation of the complex, and the trace was best fit to a double
exponential with rate constants of 2.5 and 0.5 s Dissociation of the ArsA-MgMANT-ADP Complex--
While monitoring
the MgADP induced changes in the tryptophan fluorescence of
Trp141, ArsA may predominantly report on events
occurring at the A1 NBS, studies of the changes in the fluorescence of
MANT-ADP have the potential to report on both the A1 and A2 NBS.
Consequently, the displacement of MANT-ADP bound to ArsA should resolve
both the A1 and A2 NBS if they differ in their affinities. In contrast to the ArsA-MgADP complex, dilution of the ArsA-MgMANT-ADP complex was
sufficient to induce its dissociation (Fig.
12A, trace B), with ADP apparently serving to displace more of the bound MANT-ADP (e.g. the signal increased from 11 to 24%; Fig.
12A, trace D). From a simplistic point of view
these results support our hypothesis, with MgMANT-ADP rapidly
dissociating from the low affinity site upon dilution, but MgADP
displacement needed to dissociate the MgMANT-ADP from the high affinity
site. However, the dilution might only be sufficient to dissociate
partially the ArsA-Mg·MANT-ATP complex. The dissociation of MANT-ADP
was multiphasic, and a 4-exponential equation gave the best fit,
indicative of four phases with t1/2 ~10, ~50,
and ~500 ms and ~9 s, respectively. Only the amplitudes of the
fastest two phases increased significantly when ADP was used to
dissociate the complex, and we attribute phase 1 and 2 to MANT-ADP
dissociation from the low and high affinity sites, respectively. After
dilution of the ArsA-MgMANT-ADP complex with buffer the high affinity
site would be expected to retain more MANT-ADP than the low affinity
site. Consequently, the amplitude of phase 1 would be greater than that
of phase 2 after dilution with buffer, but the amplitude of phase 2 should increase after dilution with MgADP. Consistent with this
prediction, ADP caused a larger increase in the amplitude of phase 2 (e.g. 5-fold) compared with phase 1 (e.g.
2-fold), so that the two phases had nearly equal amplitudes
(e.g. 7.9 and 7.7% fluorescence change for phases 1 and 2, respectively) and constituted 66% of the total signal. EDTA-induced
dissociation of the complex caused a small and rapid decrease in
fluorescence but was followed by an increase in fluorescence; we
attribute this behavior to the rapid dissociation of the
ArsA-MgMANT-ADP complex followed by the re-binding of MANT-ADP to form
the ArsA-MANT-ADP complex (Fig. 12A, trace
A).
If antimonite enhances the dissociation of MgADP from one site but
retards its dissociation from the other, then we might expect that the
rates of phases 1 and 2 observed for MgMANT-ADP dissociation would
increase and decrease, respectively. The dissociation of MANT-ADP from
the ArsA-MgMANT-ADP-Sb(III) complex was characterized by a 19%
decrease in MANT fluorescence that could be adequately fitted to a
triple exponential function with similar rate constants to those
determined for phases 2-4 in the absence of Sb(III) (Fig. 12A, trace C). To optimize this analysis, we
optimized the signal using a 10-fold higher concentration of ArsA, for
which the larger signal could be more clearly resolved into its
constituent phases. When 50 µM ArsA, 62.5 µM MANT-ADP, 5 mM MgCl2 was mixed
with 2.5 mM ADP, 5 mM MgCl2, the
fast phase was clearly resolved into two phases, with rate constants of
67 (± 1) and 11.0 (± 0.3) s MgADP Binding to the ArsA-Sb(III) Complex--
Since our studies
indicated that antimonite amplifies the difference in affinities of the
two NBS for MgADP, we decided to determine the kinetics of MgADP
binding to the ArsA-Sb(III) complex. The binding of (1 mM)
MgADP to the ArsA-Sb(III) complex was characterized by a multiphasic
decrease in the tryptophan fluorescence of ArsA (Fig.
13). There was a very rapid decrease in
fluorescence (e.g. phase 1, t1/2 <2 ms),
which we attribute to the binding of MgADP to the ArsA-Sb(III) complex,
followed by a slow decrease over several seconds (e.g.
phases 3 and 4, t1/2 <0.4 and 15 s,
respectively). As the Sb(III) concentration was increased we observed
the progressive appearance of a phase of intermediate rate
(e.g. phase 2, t1/2 ~10-20 ms). The
stopped-flow traces were best fit to a 4-exponential function. As shown
in Fig. 14A, for increasing
Sb(III) concentrations, an increase in the amplitude of phase 2 was
concomitant with a decrease in that for phase 4. The amplitude of phase
1 was independent of the Sb(III) concentration, as expected for a fixed
MgADP concentration, as was that of phase 3. In contrast, only the rate
constants for phases 1 and 3 had a dependence upon the Sb(III)
concentration, with those for phase 1 and 3 decreasing and increasing,
respectively, with increasing concentration (Fig. 14B).
The hyperbolic decrease in the rate of ternary complex formation
(e.g. phase 1) suggests that the ArsA alternates between conformations that differ in their affinities for Sb(III) and MgADP and
that this transition is rate-limiting for formation of the
ArsA-MgADP-Sb(III) ternary complex (30, 31). The forward and backward
rate constants for the transition and the apparent affinity for Sb(III)
can be determined from an inverse hyperbolic function (Equation 5),
If ArsA1 has a greater affinity than ArsA2
for Sb(III) (e.g. 1/K1 < 1/K6), then the binding of Sb(III) will
sequester the ArsA in the ArsA1 conformation. On the other
hand, if ArsA2-Sb(III) has a greater affinity than
ArsA1-Sb(III) for MgADP, then the binding of MgADP will
tend to sequester the ArsA in the ArsA2 conformation. The
fact that ArsA2-MgADP-Sb(III) exists implies that
1/K7 < 1/K1. If the
transition between ArsA1-Sb(III) and
ArsA2-Sb(III) is rate-limiting, then the rate of ternary
complex formation (kobs) is approximated by
Equation 5, with kf ~ k4,
kr ~ k
The hyperbolic decrease in the rate of phase 1 indicates that the ArsA
alternates between conformations that differ in their affinities for
antimonite and the nucleotide, and this behavior can be interpreted in
terms of Scheme 3, but can this scheme account for the other
phases? The rates of phases 2 and 4 are independent of the antimonite
concentration, suggesting that these are attributable to isomerizations
of the ArsA-MgADP-Sb(III) complex, whereas the rates of phases 1 and 3 are dependent upon the antimonite concentration, suggesting that they
are attributes of the binding of antimonite. One possibility is that
phase 1 is due to formation of the ArsA1-Sb(III) complex
and phase 2 to formation the ArsA1-MgADP-Sb(III) complex.
If the ArsA1-Sb(III) and ArsA2-Sb(III)
complexes have a similar fluorescence then the amplitude of phase 1 would be independent of the concentration of Sb(III), but the rate
would decrease hyperbolically because the ArsA2-Sb(III) to
ArsA1-Sb(III) transition is rate-limiting for the binding
of Sb(III). On the other hand, if the ArsA1-MgADP-Sb(III)
complex has a lower fluorescence than the ArsA-Sb(III) complexes, and
our studies indicate that it does, then the amplitude of phase 2 would
increase as the concentration of the ArsA1-Sb(III) complex
increases with the Sb(III) concentration; and the rate of
ArsA1-MgADP-Sb(III) complex formation would be
concentration-independent for a fixed concentration of MgADP.
Similarly, phases 3 and 4 can be attributed to formation of the
ArsA2-Sb(III) and ArsA2-MgADP-Sb(III)
complexes. However, the binding of Sb(III) to ArsA2 is
probably rate-limited by a slow isomerization following formation of
the ArsA2-Sb(III) complex, which is rapid but slower than
the ArsA1 to ArsA2 transition. The amplitude of
phase 4 decreases with the Sb(III) concentration as more of the ArsA is
sequestered in the ArsA1 conformation by Sb(III). Indeed,
the decrease in the amplitude of phase 4 mirrors the increase in the
amplitude of phase 2, providing strong evidence that Sb(III) pulls ArsA
between two mutually exclusive conformations. A consequence of this
interpretation is that the rate of phase 1 and the amplitudes of phases
2 and 4 should all be determined by the affinity of ArsA2
for Sb(III) (e.g. K2 in Scheme 3).
Consistent with this prediction, fits of the amplitude data for phases
2 and 4 to hyperbolic equations yielded apparent Kd
values of 200 (± 20) and 180 (± 30) µM, respectively,
which are similar to the value of 140 (± 40) µM obtained
from the rate data for phase 1 (Fig. 14). An inconsistency with this
interpretation is that in applying Equation 5, a simplifying assumption
is made that the ArsA2 state has no affinity for Sb(III),
but the rate data for phase 3, which should provide a measure of the
affinity of ArsA2 for Sb(III), indicated that this state
has moderate affinity for Sb(III) (e.g.
Kd = 200 (± 100) µM). However, the ArsA1 state need only have a higher affinity than
ArsA2 for Sb(III) to sequester the protein in this
conformation. This aside, the data clearly indicate that ArsA can adopt
different conformations that are stabilized by MgADP and Sb(III) to
different extents. We have observed similar kinetic behavior for the
binding of Mg2+ to the ArsA-ATP-Sb(III) complex, with the
rate of formation of the ArsA-ATP-Sb(III) complex decreasing in a
hyperbolic manner with increasing Sb(III) concentration (27). Although
indicative of the transition between nucleotide and
antimonite-stabilized conformations, this transition was much slower
(e.g. kf = 1 s The Pre-steady-state Production of Pi--
In
view of the fact that the present investigation indicated that the NBS
of ArsA are nonequivalent and differentially affected by antimonite, we
decided to reinvestigate the kinetics of ATP hydrolysis using a
Pi assay initiated by mixing ArsA with MgATP/Sb(III) in a
stopped-flow device (see under "Materials and Methods" for the
details of the assay). This stopped-flow experiment revealed a biphasic
burst in the production of Pi that resulted from ATP hydrolysis by ArsA. Following a lag in activity of about 40 ms, presumably attributable to the time taken for formation of the catalytic complex, there was a burst in Pi production over
the following 100 ms (e.g. k = 47 s
If product release from ArsA occurs at a faster rate than
formation of the ArsA·MgADP·Pi intermediate,
then several turnovers will occur before all the ArsA becomes trapped
in this state, after which subsequent turnovers will be rate-limited by
the dissociation of the ArsA*-MgADP·Pi complex
(e.g. k Most ATP-driven efflux pumps have a quaternary structure in which
there are two nucleotide-binding sites (NBS), both of which are
required for ATPase and transport activity. An understanding of how
these sites interact is fundamental to an understanding of the
molecular events involved in the action of these pumps. Previously, we
have undertaken a rigorous analysis of the kinetics of the binding of
the substrate ATP to the ArsA ATPase. This analysis was facilitated
using a derivative of ArsA that contains a single tryptophan residue,
Trp159, which is optically responsive to the binding of ATP
but much less so to the binding of the product ADP (28). Herein we
report on the use of another derivative of ArsA that contains a single tryptophan residue, Trp141, which is more responsive to the
binding of ADP than ATP (28). These tryptophan residues span the
DTAP region, which connects the allosteric soft metal-binding
site with the nucleotide-binding site, in the A1 half of ArsA. The
binding of MgATP to Trp159 ArsA induces a transient
increase in the tryptophan fluorescence, and we have attributed the
slow decay in fluorescence to a conformational re-arrangement of ArsA
that follows product release, and the protein adopts its original
unliganded conformation. The binding of MgADP to
Trp141 ArsA induces a quench in the tryptophan
fluorescence; and herein we report on a detailed investigation of the
kinetics of MgADP binding to Trp141 ArsA, and we
compare this with MgATP binding to Trp159 ArsA.
The initial ArsA-nucleotide complexes formed with MgATP and MgADP are
of a similar affinity (cf. 620 versus 600 µM), but overall ArsA has a higher affinity for MgATP
over MgADP (cf. 430 versus 760 µM).
In both cases, the formation of the initial complex is rate-limited by
a subsequent conformational change, but is 4 orders of magnitude faster
for the ArsA-MgADP complex. This isomerization, which may result from
closure of the NBS, will have the effect of tightening the binding of
the nucleotide, indicating apparent Kd values of 230 and 30 µM for MgATP and MgADP, respectively. We attribute
the difference between these values and the measured Kd values (e.g. 230 versus 430 µM for MgATP and 760 versus 30 µM for MgADP) to the ArsA alternating between
conformations that differ in their affinities for the Mg-nucleotide
(see kinetic Scheme 2).
The two different forms of ArsA (e.g. ArsA1 and
ArsA2 in Scheme 2) may represent open and closed
conformations of the nucleotide-binding site (NBD). This prediction is
consistent with the recently determined structure of ArsA, which
revealed MgADP locked into the A1 NBD but free to diffuse from the A2
NBD (25). This is an interesting observation because in the present
study we have found that MANT-ADP-induced dissociation of MgADP is
rapid, with a koff >223
s Antimonite affects the distribution of the two different conformational
forms of ArsA by preferentially stabilizing one conformation. Moreover,
the kinetics of the binding of MgADP to the ArsA-Sb(III) complex
strongly suggests that Sb(III) sequesters the ArsA in one of two
mutually exclusive conformations (e.g. the
ArsA1-MgADP-Sb(III) complex). Most notably, the appearance
of the phase attributed to formation of ArsA1-MgADP-Sb(III)
mirrors the disappearance of that attributed to formation of the
ArsA2-MgADP-Sb(III) complex (Fig. 14). The Sb(III)
stabilized conformation is one in which there is enhanced and retarded
release of nucleotides from the low and high affinity sites,
respectively. An obvious interpretation of these data is that Sb(III)
stabilizes the ArsA1 conformation in which the A1 NBS is
closed and the A2 NBS is open. The demonstration of both a fast and
slow burst in Pi production is consistent with two
catalytic sites of high and low affinity (Fig. 15). The site that
produces the slow burst is activated by Sb(III) so that ATP hydrolysis
at this site is no longer rate-limited by steps subsequent to the
hydrolysis step. The stabilization of the ArsA1
conformation provides a mechanism for activation by stabilizing the
open conformation of the A2 site, so that product dissociation from
this site is no longer rate-limiting.
Interestingly, we find that the binding of Sb(III) to the ArsA-MgADP
complex, and of MgATP to the ArsA-Sb(III) complex, is triphasic, and
the rate of each phase is dependent upon the antimonite concentration;
indicative of three ArsA-MgNucleotide-Sb(III) complexes, with
Kd values of about 50, 100, and 400 µM. We previously proposed that these might be slowly
inter-converting complexes, but the recent determination of the
structure of the ArsA-MgADP-Sb(III) complex has revealed that three
antimonite molecules are bound to ArsA in the complex. One Sb(III) is
bound to His148 (A1) and Ser420 (A2), one to
Cys113 (A1) and Cys422 (A2), and one to
Cys172 (A1) and His453 (A2). This suggests a
physical interpretation of the kinetic behavior; each antimonite
complex results from the addition of an antimonite molecule to ArsA as
the high, medium, and low affinity sites are successively filled.
Clearly, it is tempting to speculate on whether these antimonite
complexes play differential roles in controlling the affinities and
activities of the A1 and A2 NBDs. Mutagenesis experiments point toward
a differential role; mutation of His148 and
His453 to alanines resulted in a 5-fold reduction in the
affinity for metal-stimulated ATP
hydrolysis,6 whereas
significantly more severe were mutations of the cysteine ligands. The
Cys113 The structure and function of ArsA are suggestive of the following
model of the events occurring at the NBS. In the absence of ligands,
ArsA oscillates between two conformations as follows: ArsA1
in which the A1 site is closed and the A2 site open, and
ArsA2 in which the A1 site is open and the A2 site closed.
MgATP can bind to either conformation with the distribution of
conformers dependent upon the affinities of the A1 and A2 sites.
However, the binding of Sb(III) to the high affinity antimonite-binding site sequesters the ArsA into the ArsA1 conformation. This
triggers a burst in MgATP hydrolysis at the A1 site, catalyzing the
build up of a pre-steady-state intermediate that rate-limits further
turnover. Since Sb(III) hinders the dissociation of MgADP from the high
affinity NBS, this suggests that the intermediate is ArsA-MgADP. Why
should Sb(III) activate the A1 NBS? One possibility is that the
hydrolysis of MgATP during the burst phase is required to drive a
conformational change in the ArsA. This conformational change could
involve an enhancement in the interaction of the A1 and A2 domains,
possibly leading to formation of the intermediate antimonite-binding
site. Similarly, the binding of Sb(III) to this site may be necessary
to drive a further conformational change, activating the A2 site so
that it can catalyze the rapid hydrolysis of MgATP, leading to a
further tightening of the A1-A2 domain interaction and formation of the
low affinity antimonite-binding site. The binding of Sb(III) to this
site triggers the rapid release of product MgADP. In other words, the
three Sb(III) ions "zip" the A1 and A2 domains together,
`locking' ArsA in the activated form, in which there is continuous
rapid hydrolysis of MgATP at the A2 NBS. This must be coupled to the
pumping of Sb(III) molecules across the membrane, but how?
The structure of ArsA indicates that the buried surface between the A1
and A2 domains is relatively small compared with the total surface of
the enzyme, such that ArsA is essentially a hollow protein with a large
central cavity (25). The metal-binding site is open below this
central cavity, and it is sealed above by the loop between helices H9
and H10 that provide the ligand Cys172. Thus, it is
possible that during the catalytic cycle of ArsA antimonite ions may
access the metal site from the central cavity, to then be injected into
ArsB in association with a conformational change in the H9-H10 region
(33). ArsA would then be similar to a "pumping heart" that expands
while drawing antimonite ions from the cytoplasm and contracts while
expelling them into the membrane channel provided by ArsB. To achieve
this pump action, the ArsA must bind and release Sb(III) ions into the
ArsB channel. The hydrolysis of MgATP at the A2 site may drive the ArsA
between conformations that have high and low affinity for antimonite. In this context, we note that the affinity of ArsA for Sb(III) is
increased by the binding of Mg·nucleotides. Could the three antimonite sites be used to relay the Sb(III) ion through ArsA to ArsB?
The release of MgADP from the A2 NBS could trigger a reduction in the
affinity of the "high affinity" binding site for Sb(III), allowing
its release to ArsB, but this site can only be refilled by Sb(III) from
the intermediate affinity site, and the intermediate site by Sb(III)
from the low affinity site, thus providing a mechanism to draw Sb(III)
into ArsA and eject it into the ArsB channel. In effect, the two
Pi burst phases are required to "prime the pump" with Sb(III).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices and a nucleotide binding domain (NBD).
-helices arranged in two groups of six, and their NBDs contain allosteric sites. As such, the arsenical
pump is a useful model for the study of the molecular mechanism of
resistance pumps. However, the arsenical pump is composed of two
proteins, ArsA and ArsB; ArsA is the 63-kDa catalytic subunit that
couples ATP hydrolysis to oxyanion translocation, whereas ArsB is the
45-kDa membrane sector of the pump, a 12-helix protein that acts as the
oxyanion-translocating sector of the pump. ArsA is normally bound to
ArsB but can be purified as a soluble ATPase in the absence of ArsB,
facilitating detailed studies of its structure-function. 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 25-residue linker (residues 283-320) (19), and
each domain has a consensus NBD. Site-directed mutagenesis of these
sequences indicates that both NBDs are required for both catalysis and
resistance (20, 21). On the basis of genetic studies, the A1 and A2
NBDs have been shown to interact during catalysis (22, 23) and exhibit
strong positive cooperativity (24). The ArsA ATPase is allosterically
regulated by its substrate metalloids As(III) and Sb(III) (1). This is
similar to the allosteric activation of the P-glycoprotein by drug
substrates (1) and may be a common feature of ABC transporters. Indeed,
bacterial ABC transporters frequently include ATPase subunits with an
allosteric site that controls the ATPase activity (4). In a most
important advance, the structure of ArsA has been determined to atomic
resolution (25). The structure of ArsA indicates that the A1 and A2
halves of ArsA are arranged into two domains, with the NBS formed from residues contributed from both domains and are located at the interface
between them in close proximity to one another. These structural and
functional properties of ArsA indicate that the NBS are likely to
resemble those in P-glycoprotein and certainly more so than HisP. In
common with P-glycoprotein, the NBD of ArsA are in close proximity,
face one another, and interactions between them are necessary to
support ATPase activity and transport.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C. The
concentration of purified ArsA was determined by UV absorbance at 280 nm. The extinction coefficients for W141H6 ArsA and W159H6 ArsA were
calculated to be 21,530 and 20,250 M
1 cm
1,
respectively (28).
kt;
where s represents the change in signal (e.g.
volts or % fluorescence), t the time, A and
k the amplitude and rate constant for the signal change,
respectively) or multiple exponential functions (e.g. s = A1·exp
k (1)t + A2·exp
k (2)t + A3·exp
k (3)t, for a triple
exponential function) using the nonlinear regression software with the
Applied Photophysics stopped-flow. As a guide to the adequacy of a
particular equation to define a data set, we increased the number of
exponential parameters within the equation until the data points were
randomly distributed about the best fit line in a plot of the residual
variance. 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.
E360 = 12 mM
1·cm
1.
For stopped-flow studies, both syringes contained the components of the
reaction kit, but one syringe contained 10 µM ArsA and the other 1 mM MgATP.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
MgADP-induced conformational changes in
ArsA. In the upper panel two semi-logarithmic plots of
stopped-flow traces generated by mixing 5 µM ArsA with 1 mM ADP, 5 mM MgCl2 (trace
A) and 5 µM ArsA with 0.05 mM ADP, 5 mM MgCl2 (trace B) are shown.
Changes in the ArsA fluorescence were recorded with Ex = 292.5 nm
and Em >335 nm, and one vertical division represents a fluorescence
change of 5%. The smooth curve through each trace is the best fit to a
4-exponential equation with rate constants of 510 (± 40), 0.14 (± 0.02), 0.030 (± 0.002), and 0.0020 (± 0.0001)
s 1 (trace A); and 84 (± 4), 0.20 (± 0.02), 0.021 (± 0.001), and 0.0020 (± 0.0001)
s
1 (trace B). In the
lower panel, trace A is plotted on a linear time scale, with
the first 50 ms of the reaction shown as an inset. The
smooth curve through the trace is the best fit to the 4-exponential
equation defined for trace A in the top panel.
Clearly, the fitted equation provides an adequate fit of the data at
both the early and late data points, as indicated by the best fit line
and, as shown in the bottom panel, a plot of the residual
variance of the data about the best fit curve. One vertical division
represents a fluorescence change of 2.5% and 1.25% for the
inset.
indicated maximal (kmax) and minimal
(kmin) rates of binding of 600 (± 20) and 30 (± 20) s
(Eq. 1)
1, respectively, and a
Kd of 600 (± 100) µM. This behavior is consistent with the fast phase attributable to a two-step binding process shown in Scheme 1,
where the first step is a rapid equilibrium binding of MgADP to
ArsA, followed by a rate-limiting isomerization of the ArsA-MgADP complex. Applying Equation 1 to Scheme 1, Kd is
equivalent to 1/K2 (=
k
2/k2),
kmax = k3 + k
3 and kmin = k
3. The overall equilibrium constant is a
function of both K2 and K3 (Equation 2),
where K2 = 0.6 mM and
K3 = k3/k
(Eq. 2)
3 = 600/30 = 20. Thus, the apparent Kd value can be calculated as
24 µM. However, a plot of the amplitude data (Fig.
2B), which will provide a measure of the overall
Kd, yielded a value of 800 (± 100)
µM. A plausible explanation for this behavior is that the
ArsA exists in two rapidly inter-converting conformational forms that
differ in their affinities for MgADP (Scheme 2),
The overall Kd value is a function of
K1, K2, and
K3 (Equation 3),
Accordingly, K1 can be calculated, from the
overall Kd and K2, as 27, indicating that 96% of the ArsA is in the ArsA1
conformation prior to the binding of MgADP.
(Eq. 3)
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Fig. 2.
The concentration dependence of the rate of
binding of MgADP to ArsA. A series of stopped-flow records were
generated by mixing ArsA with 5 mM MgCl2 and
ADP, at the indicated concentration, in a stopped-flow device. The rate
(A) and amplitude (B) of the fluorescence signal
for the binding of MgADP to ArsA are plotted as a function of the ADP
concentration. The curves through the data points are the best fits to
a hyperbolic equation, indicating values of 600 (± 20)
s 1, 30 (± 20) s
1,
and 600 (± 100) µM for kmax,
kmin, and Kd, respectively,
in A, and of 800 µM (± 100) and 26% (± 1)
for the Kd and maximal fluorescence change
(Fmax) in B.
1 for 125 µM MANT-ATP (Fig.
3B)).2 In
contrast, extrapolation of the data in Fig. 2 to the y axis suggested a much slower rate of dissociation (e.g.
k = 30 (± 20) s
1).
Conceivably, this might provide a measure of
k
3 rather than k
2 in
Scheme 1, but it could also arise if the two NBS had different
affinities (see below).
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Fig. 3.
The dissociation of the ArsA-MgADP complex
induced by MANT-ADP displacement. Three semi-logarithmic plots of
stopped-flow traces generated by mixing 5 µM ArsA, 0.5 mM ADP, 5 mM MgCl2 with 0.125 mM MANT-ADP, 5 mM MgCl2
(A); 5 µM ArsA, 5 mM
MgCl2 with 0.5 mM ADP, 0.125 mM
MANT-ADP, 5 mM MgCl2 (B); and 5 µM ArsA, 5 mM MgCl2 with 0.125 mM MANT-ADP, 5 mM MgCl2
(C) are shown. Changes in the ArsA fluorescence were
recorded with Ex = 292.5 nm and Em > 420 nm, and one
vertical division represents a fluorescence change of 2.5%. The smooth
curve through traces A and B are the best fits to
a 3-exponential equation with rate constants of 36 (± 1), 1.9 (± 0.1), and 0.09 (± 0.01) s 1 (A)
and 22 (± 2), 7.8 (± 0.3), and 0.094 (± 0.005)
s
1 (B).
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Fig. 4.
Mg2+-induced conformational
changes in the ArsA-ADP complex. A semi-logarithmic plot of a
stopped-flow trace generated by mixing 5 µM ArsA, 0.5 mM ADP with 5 mM MgCl2. Changes in
the ArsA fluorescence were recorded with Ex = 292.5 nm and Em > 335 nm, and one vertical division represents a fluorescence change
of 2.5%. The smooth curve through each trace is the best fit to a
4-exponential equation with rate constants of 139 (± 3), 2.3 (± 0.3),
0.050 (± 0.003), and 0.0020 (± 0.0001)
s 1.
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Fig. 5.
The ADP concentration dependence of the rate
of formation of the ArsA-MgADP complex induced by the binding of
Mg2+ to the ArsA-ADP complex. A series of stopped-flow
records were generated by mixing ArsA, equilibrated with ADP at the
indicated concentration, with 5 mM MgCl2 in a
stopped-flow device. The rate (A) and amplitude
(B) of the fluorescence signal for the formation of the
ArsA-MgADP complex are plotted as a function of the ADP concentration.
The curves through the data points are the best fits to a hyperbolic
equation, indicating values of 170 (± 10) s 1
and 90 (± 10) µM for kmax and
Kd, respectively, in A, and of 190 µM (± 20) and 29% (± 1) for the Kd
and maximal fluorescence change (Fmax) in
B.
Thus, the fit yielded a Kd of 90 (± 10)
µM and a maximal rate of binding of 170 (± 10)
s
(Eq. 4)
1. This analysis indicated that the back
rate, determined from the intercept of the y axis, was too
slow for accurate determination. A possible explanation for the
relatively high affinity binding might be that ADP alone stabilizes the
ArsA2 conformation. Consistent with this prediction, the
amplitude data indicated a Kd of 190 (± 20)
µM and a maximal quench in fluorescence
(Fmax) of 29 (± 1)% (Fig. 5B).
1), as compared with that from ArsA and
MgADP (e.g. kmax = 600 (± 100)
s
1), indicates a more complex binding process
than the simple addition of Mg2+ to the ArsA-ADP complex.
The Mg2+ cannot bind directly to the ArsA2-ADP
complex, nor can complex formation be rate-limited by ADP dissociation
from ArsA2, otherwise the rate of complex formation would
be independent of the ADP concentration. A plausible explanation of
this kinetic behavior is that at sufficiently high concentrations of
ADP, when the binding of MgADP would be faster than the dissociation of ADP from the ArsA2-ADP complex, ADP dissociation becomes
rate-limiting. Thus interpreting the data implies that ADP dissociates
from the ArsA-ADP complex with a rate constant of 170 s
1.
1 for the fast phase
(Fig. 6A). In contrast, when
ArsA was preincubated with ADP, before mixing with MANT-ADP, only the
rapid increase in MANT fluorescence was apparent, although the data
were best fit to a double exponential function, with the fast phase
occurring with a rate constant of 170 (± 40)
s
1 (Fig. 6B). It is most likely
that the difference in reaction profiles is attributable to
dissociation of the ArsA-ADP complex rate-limiting the binding of
MANT-ADP. A proposal that was supported by the fact that a similar
trace to that obtained for the binding of MANT-ADP alone was obtained
when ArsA was mixed with MANT-ADP/ADP (Fig. 6C). Although,
as expected, preincubating the MANT-ADP with ADP caused a reduction in
the signal amplitude and yielded a slower rate constant, this is
consistent with the ADP competing with the MANT-ADP for binding to
ArsA. We conclude that ADP dissociates from ArsA with a rate constant
of 170-200 s
1. This conclusion is further
supported by the fact that reducing the ADP concentration with which
the ArsA was preincubated from 0.5 to 0.25 mM had no effect
upon the apparent rate constant for the binding of MANT-ADP (data not
shown).
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Fig. 6.
The dissociation of the ArsA-ADP complex
induced by MANT-ADP displacement. Three semi-logarithmic plots of
stopped-flow traces generated by mixing 5 µM ArsA with
62.5 µM MANT-ADP (A), 5 µM ArsA,
0.5 mM ADP with 62.5 µM MANT-ADP
(B), and 5 µM ArsA with 0.5 mM
ADP, 62.5 µM MANT-ADP (C) are shown. Changes
in the ArsA fluorescence were recorded with Ex = 292.5 nm and
Em > 420 nm, and one vertical division represents a fluorescence
change of 2.5%. The smooth curve through traces
A and C is the best fit to a 4-exponential
equation with rate constants of 400 (± 100), 24 (± 1), 0.73 (± 0.03), and 0.045 (± 0.001) s 1
(A); and 350 (± 10), 24 (± 1), 0.83 (± 0.03), and 0.046 (± 0.001) s
1 (C). The smooth
curve through trace B is the best fit to a double
exponential equation with rate constants of 170 (± 40) and 14.8 (± 0.4) s
1, where the fast phase represents 78%
of the total signal amplitude.
1, and relative signal amplitudes of
0.9 and 0.1, respectively, giving a total fluorescence change of 22%.
This value for the increase in fluorescence is comparable to the
decrease in fluorescence for the binding of Mg2+ to the
ArsA-ADP (e.g. 5 µM ArsA/0.5 mM
ADP) complex (cf. 22 versus 21%), indicating
that the 20 mM EDTA caused the ArsA-MgADP complex to fully
dissociate.3 The dominant
phase, which occurred with a rate constant of 6.6 s
1, must be attributable to the dissociation
of the complex because there are no isomerizations of a comparable rate
for ternary complex formation from ArsA/ADP and Mg2+
(e.g. see Fig. 4). The slow rate of dissociation suggests
that the ArsA-MgADP complex is relatively stable. Indeed, dilution of
the ArsA-MgADP complex, by mixing with buffer in the stopped-flow, was
insufficient to dissociate the complex (Fig. 7E). These data raise an apparent anomaly because the binding of MgMANT-ADP to ArsA is
not rate-limited by the dissociation of the ArsA-MgADP complex
(e.g. see Fig. 3). A simple explanation for this behavior is
that the A1 and A2 NBS have different affinities. EDTA is necessary to
induce dissociation of MgADP from the high affinity site, but MANT-ADP
can displace MgADP from the low affinity site. Since the tryptophan
residue that acts as the reporter of the EDTA effects is positioned in
the A1 domain, this suggests that the A1 site has greater affinity than
the A2 site.
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Fig. 7.
EDTA-induced dissociation of the ArsA-MgADP
and ArsA-MgADP-Sb(III) complexes. A set of semi-logarithmic plots
of stopped-flow traces generated by mixing 5 µM ArsA, 0.5 mM ADP, 5 mM MgCl2 with 100 mM EDTA (A); 5 µM ArsA, 0.5 mM ADP, 5 mM MgCl2 with 20 mM EDTA (B); 5 µM ArsA, 0.5 mM ADP, 5 mM MgCl2, 0.1 mM Sb(III) with 20 mM EDTA (C); 5 µM ArsA, 0.5 mM ADP, 5 mM
MgCl2, 2.5 mM Sb(III) with 20 mM
EDTA (D); and 5 µM ArsA, 0.5 mM
ADP, 5 mM MgCl2 with buffer (E) are
shown. Changes in the ArsA fluorescence were recorded with Ex = 292.5 nm and Em > 335 nm. One vertical division represents a
fluorescence change of 5%. The smooth curves through traces
B and C are the best fits to a double exponential
equation with rate constants of 6.60 (± 0.02) and 0.170 (± 0.004)
s 1 (B); and 2.50 (± 0.03) and
0.48 (± 0.01) s
1 (C). The data
imply that Sb(III) retards the dissociation of the ArsA-MgADP-Sb(III)
complex. Note that traces A and E are displaced
from the other for clarity of presentation.
1, which were
independent of the Sb(III) concentration (data not shown). However, a
very rapid increase in fluorescence was noted for the higher
concentrations of Sb(III) used, which might be due to the binding of
Sb(III), followed by three slow isomerizations of the ArsA-Sb(III)
complex. A hyperbolic fit of the total amplitude of the signal
indicated a Kd of 540 µM and a maximal quench of 5% (data not shown), indicating that Sb(III) is bound with
low affinity in the absence of nucleotides (see below).
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Fig. 8.
Antimonite-induced conformational changes in
ArsA and the ArsA-MgADP complex. A set of semi-logarithmic plots
of stopped-flow traces generated by mixing 5 µM ArsA with
2.5 mM Sb(III) (A); 5 µM ArsA, 1 mM ADP, 5 mM Mg2+ with 1.5 mM Sb(III) (B); and 5 µM ArsA, 1 mM ADP, 5 mM Mg2+ with 50 µM Sb(III) (C), are shown. Changes in the ArsA
fluorescence were recorded with Ex = 292.5 nm and Em > 335 nm. One vertical division represents a fluorescence change of 2.5%.
The smooth curve through each trace is the best fit to a 3-exponential
equation with rate constants of 21 (± 1), 0.31 (± 0.02), and 0.031 (± 0.003) s 1 (A); 89 (± 1), 3.2 (± 0.1), and 0.08 (± 0.01) s
1
(B); and 39.1 (± 0.4), 1.20 (± 0.03), and 0.044 (± 0.004)
s
1 (C).
F <5%), followed by a
slow decay in fluorescence over several minutes (Fig. 8B).
Sb(III) did not reverse the quench in tryptophan fluorescence caused by
the binding of MgADP to ArsA, and we attribute the decrease in
fluorescence to the formation of an ArsA-MgADP-Sb(III) complex.
Furthermore, as discussed below, EDTA can reverse the quench in
fluorescence of this complex, confirming its identity as the
ArsA-MgADP-Sb(III) complex. For most stopped-flow traces, the data were
adequately fitted to a triple exponential function (e.g.
Fig. 8C), but as the Sb(III) concentration was increased
there was a deviation of the early part of the trace from this function
(e.g. Fig. 8B). This is suggestive of the
presence of a faster phase, but since this was not well resolved all
the traces were fitted to triple exponential functions. As indicated by
Fig. 8, B and C, the most pronounced change, with
the Sb(III) concentration, was in the rate and amplitude of the fast
phase, and we attribute this phase to the formation of the
ArsA-MgADP-Sb(III) complex. The rate of complex formation increased in
a hyperbolic manner, and the data were best fit to a hyperbolic
equation (e.g. Equatrion 1), yielding values for
kmax, kmin, and
Kd of 80 (± 10) s
1, 3 (± 1) s
1, and 60 (± 20) µM,
respectively (Fig. 9A).
Applying Equation 2 yielded a value of 2 µM for the
overall Kd. On the other hand, a fit of the
amplitude data (Fig. 9B) to a hyperbolic equation yielded an
overall Kd of 30 (± 3)
µM,4 suggesting
that the ArsA-MgADP complex exists in equilibrium between forms that
have high and low affinity (e.g. K1 = 10) for Sb(III). This is perhaps not surprising because the unliganded ArsA exists in two forms that differ in their affinity for MgADP. Although less pronounced, the rates of the intermediate and slow phases
increased in a hyperbolic manner with the antimonite concentration, indicating Kd values of 200 (± 100) and 300 (± 100) µM, respectively (data not shown). Previously, we
noted that the binding of MgATP to the ArsA-Sb(III) complex caused a
triphasic quench in the tryptophan fluorescence of
Trp159 ArsA; and the rates of the three phases were
dependent upon the antimonite concentration, increasing with
Kd values of 50 (± 10), 100 (± 30), and 400 (± 100) µM (27).
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Fig. 9.
The antimonite concentration dependence of
the rate and amplitude of the signal associated with formation of the
ArsA-MgADP-Sb(III) complex due to the binding of Sb(III) to the
ArsA-MgADP complex. A series of stopped-flow records were
generated by mixing ArsA, equilibrated with 1 mM ADP, 5 mM MgCl2, with Sb(III), at the indicated
concentration, in a stopped-flow device. The rate (A) and
amplitude (B) of the fluorescence signal for the formation
of the ArsA-MgADP-Sb(III) complex are plotted as a function of the
antimonite concentration. The curves through the data points are the
best fits to a hyperbolic equation, indicating values of 80 (± 10)
s 1, 3 (± 1) s
1,
and 60 (± 20) µM for kmax,
kmin, and Kd, respectively,
in A; and of 30 µM (± 3) and 5.7% (± 0.1)
for the Kd and maximal fluorescence change
(Fmax) in B.
1; kmin = ~1
s
1, and Kd = 330 µM) (27). EDTA was also able to reverse the enhanced
fluorescence of this pre-steady-state intermediate, with a rate
constant of 4 s
1, confirming that the
intermediate was an ArsA-Mg·nucleotide complex that could be
destabilized by withdrawing Mg2+ (27). Consistent with this
interpretation, pre-equilibrating the Trp159 ArsA
with Sb(III), to form the ArsA-Sb(III) complex, prevented formation of
the transient with enhanced fluorescence upon the addition of MgATP.
However, in the present investigation with Trp141
ArsA, Sb(III) did not reverse the decrease in fluorescence that occurs
upon formation of the ArsA-MgADP complex but caused about a further 6%
decrease in fluorescence, indicative of the formation of the
ArsA-MgADP-Sb(III) complex. This behavior suggests that Sb(III)
specifically destabilizes the ArsA-MgADP·Pi complex.
1 (data not shown).
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Fig. 10.
The binding of Mg2+ to the
ArsA-ADP-Sb(III) complex. A set of semi-logarithmic plots of
stopped-flow traces generated by mixing 5 mM
Mg2+ with 5 µM ArsA, 1 mM ADP,
equilibrated with 0 µM Sb(III) (A), 25 µM Sb(III) (B), and 2500 µM
Sb(III) (C) are shown. Changes in the ArsA fluorescence were recorded
with Ex = 292.5 nm and Em > 335 nm. One vertical division
represents a fluorescence change of 2.5%. The smooth curve through
each trace are the best fits to 4-exponential equations yielding rate
constants of 460 (± 30), 142 (± 2), and 0.40 (± 0.03), and 0.027 (± 0.003) s 1 (A); 270 (± 10), 71 (± 2), 2.0 (± 0.1), and 0.067 (± 0.003) s
1
(B); and 269 (± 4), 30 (± 1), 2.0 (± 0.1), and 0.049 (± 0.002) s
1 (C). The measured
fluorescence changes were 23 (A), 25 (B), and
22% (C).
1, respectively (cf. 2.5 versus 7 s
1). Although the total
signal amplitude was almost equivalent to that in the absence of
Sb(III) (cf. 20 versus 22%), the relative proportions of the two phases had changed to 0.65 and 0.35 for the fast
and slow phases, respectively (cf. 0.9 and 0.1, respectively, for the ArsA-MgADP complex, Fig. 7B). Clearly,
the implication is that Sb(III) retards dissociation of
Mg2+ from the ArsA-MgADP complex. However, we found that
the signal amplitude was reduced in the presence of Sb(III). To
investigate this phenomena more fully, the experiment was repeated for
a series of Sb(III) concentrations. Although the rate constants for the two phases remained constant with increasing antimonite concentrations (e.g. phase 1 and 2 varied between 2 and 3 and 0.5-1.5
s
1, respectively; data not shown), the signal
amplitude decreased from 22 to 8% in a hyperbolic manner, with a
Kd of 260 (± 30)
µM5 (Fig.
11A). Although
the binding of Sb(III) to the ArsA-MgADP complex induces a quench in
the tryptophan fluorescence of the complex, this is less than 6%.
Moreover, the binding of Mg2+ to the ArsA-ADP and
ArsA-ADP-Sb(III) complexes produced a similar quench in protein
fluorescence (see Fig. 10). The reduced signal amplitude for the
EDTA-induced dissociation of the ArsA-MgADP complex in the presence of
Sb(III) cannot simply be accounted for by formation of the
ArsA-ADP-Sb(III) complex, with reduced fluorescence, at the end of the
reaction. A simple interpretation of this behavior is that the
ArsA-MgADP-Sb(III) complex has less MgADP bound at the start of the
reaction than the ArsA-MgADP complex. This is probably due to
antimonite acting differentially at the two nonequivalent NBS of ArsA,
triggering release of MgADP from one site but retarding release from
the other site. Consistent with this interpretation, Fig.
11A establishes that in the presence of Sb(III) the
reduction in signal is attributable to a decrease in the amplitude of
the fast phase, rather than an equal reduction in both the fast and
slow phases. If the fast and slow phases were attributed to the
dissociation of MgADP and a subsequent conformational change in the
unliganded ArsA, respectively, then a reduction in the amount of bound
MgADP would, as observed, only decrease the amplitude of the fast
phase.
View larger version (13K):
[in a new window]
Fig. 11.
The antimonite concentration dependence of
the EDTA-induced dissociation of the ArsA-MgADP-Sb(III) complex. A
series of stopped-flow records were generated by mixing by mixing 5 µM ArsA, 0.5 mM ADP, 5 mM
MgCl2, equilibrated with the indicated concentrations of
Sb(III), with 20 mM EDTA in a stopped-flow device. These
traces were fitted to a double exponential equation, and in the
upper panel, the amplitudes of the fast ( ) and slow (
)
phases and the total amplitude (
) are plotted as a function of the
antimonite concentration. For comparison, the amplitudes of the
corresponding phases for the dissociation of the ArsA-MgADP complex in
the absence of Sb(III) are shown on the plot as the large
symbols
,
,
, respectively. The curve through the data
points for the decrease in total amplitude is the best fit to a
hyperbolic equation, indicating that the fluorescence decreases from 22 (± 1) to 8.0% (± 0.3) with a Kd 260 µM (± 30). This decrease in fluorescence is clearly
attributable to a decrease in the fast phase, with the slow phase
remaining constant. In the lower panel the corresponding
rate constants for the fast (
) and slow (
) phases are
shown.
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Fig. 12.
Dissociation of the ArsA-MgMANT-ADP
complex. A, five semi-logarithmic plots of stopped-flow
traces generated by mixing 5 µM ArsA, 25 µM
MANT-ADP, 5 mM MgCl2 with 20 mM
EDTA (trace A); 5 µM ArsA, 25 µM
MANT-ADP, 5 mM MgCl2 with buffer (trace
B); 5 µM ArsA, 25 µM MANT-ADP, 5 mM MgCl2, 0.5 mM Sb(III) with 2.5 mM ADP, 5 mM MgCl2 (trace
C); 5 µM ArsA, 25 µM MANT-ADP, 5 mM MgCl2 with 2.5 mM ADP, 5 mM MgCl2 (trace D); and 5 µM ArsA, 5 mM MgCl2 with 25 µM MANT-ADP, 2.5 mM ADP, 5 mM
MgCl2 (trace E) are shown. Changes in the ArsA
fluorescence were recorded with Ex = 292.5 nm and Em > 420 nm, and one vertical division represents a fluorescence change of
2.5%. The smooth curve through trace D is the
best fit to a 4-exponential equation with rate constants of 71 (± 2)
s 1 (7.9% fluorescence change), 13.0 (± 0.4)
s
1 (7.7% fluorescence change), 1.50 (± 0.04) s
1 (4.8% fluorescence change), and
0.060 (± 0.001) s
1 (3.4% fluorescence
change). The curve through trace B is a fit to a
4-exponential equation in which the rate constants were held constant
at the values derived from trace D, so as to
allow a comparison of the amplitudes of the four phases in each trace.
This procedure yielded amplitudes of 3.5, 1.6, 3.2, and 2.4% for
phases 1-4, respectively, for trace B. Trace C could be
adequately fitted by a triple exponential function, with rate constants
of 14.0 (± 0.3), 1.10 (± 0.02), and 0.080 (± 0.001)
s
1, as shown by the smooth curve through the
trace, indicating the absence of fast phase 1. B, two
semi-logarithmic plots of stopped-flow traces generated by mixing 50 µM ArsA, 62.5 µM MANT-ADP, 5 mM
MgCl2 with 2.5 mM ADP, 5 mM
MgCl2 (A); and 50 µM ArsA, 62.5 µM MANT-ADP, 5 mM MgCl2, 0.5 mM Sb(III) with 2.5 mM ADP, 5 mM
MgCl2 (B). The smooth curve through trace
A is the best fit to a 4-exponential equation with rate constants
of 67 (± 1), 11.0 (± 0.3), 1.30 (± 0.03), and 0.080 (± 0.002)
s
1. Trace B could be adequately
fitted by a triple exponential function, with rate constants of 12.0 (± 0.2), 0.60 (± 0.01), and 0.050 (± 0.001)
s
1, as shown by the smooth curve through the
trace, indicating the absence of fast phase 1.
1 (Fig.
12B, trace A). In the presence of antimonite the faster of
the two phases was clearly absent, and the slower phase occurred with a
rate constant of 12.0 (± 0.2) s
1 (Fig.
12B, trace B). The effect of antimonite was to
abolish the fast phase, and we attribute this behavior to
Sb(III)-induced dissociation from the low affinity site. Although
complicated by the multiphasic traces, these results are highly
consistent with our conclusion that the ArsA NBS are nonequivalent, one
site allowing rapid release of nucleotides and the other slow release, and that antimonite acts differentially on these sites causing product
dissociation from the low affinity site.
View larger version (14K):
[in a new window]
Fig. 13.
The binding of MgADP to the ArsA-Sb(III)
complex. A set of semi-logarithmic plots of stopped-flow traces
generated by mixing 5 µM ArsA, 1 mM ADP, 5 mM Mg2+ with 25, 100, 150, 200, 375, and 750 µM Sb(III) are shown in A. The traces are
arranged from top to bottom in order of increasing Sb(III)
concentration, with the top trace corresponding to the lowest Sb(III)
concentration. Changes in the ArsA fluorescence were recorded with
Ex = 292.5 nm and Em > 335 nm. One vertical division
represents a fluorescence change of 2.5%. The smooth curve through the
traces generated with 25 µM (top trace) and
750 µM (bottom trace) Sb(III) are the best
fits to 3- and 4-exponential equations in B and
C, respectively. For the 3-exponential analyses in
B, there is clearly a systematic deviation of the fitted
curves about the measured curves indicate the inadequacy of this fit to
the data. The 4-exponential analyses in C yielded rate
constants of 740 (± 20), 58 (± 5), 0.770 (± 0.002), and 0.005 (± 0.001) s 1 for 25 µM Sb(III);
and 460 (± 12), 46 (± 1), 2.060 (± 0.003), and 0.005 (± 0.001)
s
1 for 750 µM Sb(III).
View larger version (16K):
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Fig. 14.
The antimonite concentration dependence rate
and amplitude of the signal associated with formation of the
ArsA-MgADP-Sb(III) complex due to the binding of MgADP to the
ArsA-Sb(III) complex. A series of stopped-flow records were
generated by mixing ArsA, equilibrated with the indicated
concentrations of Sb(III), with 1 mM ADP, 5 mM
MgCl2 in a stopped-flow device. These traces were fitted to
a 4-exponential equation, and the amplitude (A) and log of
the rate (B) for each phase are plotted as a function of the
antimonite concentration; the symbols used for phases 1-4 are ,
,
, and
, respectively. The curves through the phase 2 (
)
and 4 (
) data points in A are the best fits to hyperbolic
equations, indicating Kd values of 200 (± 20) and
180 (± 30) µM, respectively. The curves through the
phase 1 (
) and 3 (
) data points in B are the best fits
to hyperbolic equations (e.g. Equations 5 and 1,
respectively), indicating values for kmax (or
kf), kmin (or
kr), and Kd of 490 (± 10)
s
1, 310 (± 20) s
1,
and 140 (± 40) µM; and 1.7 (± 0.2)
s
1, 0.5 (± 0.2)
s
1, and 200 (± 100) µM,
respectively.
A fit of the phase 1 rate data (
(Eq. 5)
) in Fig. 14B to
Equation 5 indicated values for kf,
kr, and Kapp of 490 (± 10)
s
1, 310 (± 20) s
1,
and 140 (± 40) µM, respectively. The simplest mechanism
consistent with this kinetic behavior would be one in which Sb(III) and
MgADP compete for binding to ArsA. However, as discussed above, Sb(III) does not reverse the quenched fluorescence of the ArsA-MgADP complex, but subsequent treatment with EDTA does reverse the quench,
indicating the formation of an ArsA-MgADP-Sb(III) ternary
complex. Moreover, recent crystallographic studies have
revealed the existence of the ArsA-MgADP-Sb(III) ternary
complex (25). A more elaborate model of ternary complex
formation is required, and we propose the following Scheme
3:
View larger version (10K):
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Scheme 3.
4, and
Kapp ~ K2. In the
absence of Sb(III) ArsA exists as an equilibrium mixture of 61%
ArsA1-MgADP and 39% ArsA2-MgADP.
1
and kr = 0.5 s
1), and the
antimonite favored conformation had a higher affinity of 65 µM for antimonite (27).
1) and a second burst was apparent over
10 s (e.g. k = 0.2 s
1). Both bursts were of a similar amplitude,
with the production of 6 and 7 µM Pi per
µM of ArsA, indicating that the ArsA turns over several
times before the steady-state intermediate builds up to a rate-limiting
concentration. In contrast, ArsA equilibrated with Sb(III) produced a
fast burst, with a similar rate constant and amplitude, but the slow
burst phase was absent, being replaced by an apparent lag in activity
that was most pronounced during the first 2 s of the reaction
(Fig. 15A, trace
B). Furthermore, when Sb(III) was omitted from the reaction, there
was no fast burst, and the slow burst was not readily apparent over
this 100-s time base (Fig. 15B, trace C).
Previously, in the absence of Sb(III), we noted a slow burst in
Pi release of about 2 nmol
Pi·nmol
1 ArsA, which occurred
over 2000 s, with a rate constant of 0.001 s
1 (26). This behavior indicates that
antimonite rapidly activates both NBS, so that they can hydrolyze
MgATP, with several molecules undergoing hydrolysis in a burst of
activity. In our previous analysis of the ATPase mechanism, we
postulated that following product release, a conformational transition
of the ArsA back to its original conformation was rate-limiting for
steady-state ATP hydrolysis in the absence of Sb(III). The present
study suggests more complex behavior because several turnovers are
necessary to allow the build up of an ArsA conformation that
rate-limits the steady state. An interesting possibility, which has
been proposed for other ATPases (31), is the trapping of an
ArsA·MgADP·Pi intermediate (Scheme 4),
View larger version (18K):
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Fig. 15.
Phosphate burst phase of ArsA ATPase.
10 µM ArsA was mixed with 1 mM ATP in a
stopped-flow device, 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. This experiment was performed in the presence of 0.5 mM Sb(III), equilibrated with either the ATP (traces
A) or ArsA (traces B), or in the absence of Sb(III)
(traces C). A and B show the
production of Pi over the first 140 ms and 100 s of
the reaction, respectively. Two bursts in Pi production,
over 100 ms and 20 s, could be resolved when the ArsA was
simultaneously mixed with ATP and Sb(III); and thereafter
Pi production was linear over the next 80 s, but
extended analyses established that Pi production remained
linear over 1000 s. The data between 40 and 250 ms were fitted to
a single-exponential function, and the data between 0.25 and 90 s
were fitted to a single exponential function with a linear term. This
analysis indicated that the fast and slow burst phases occurred with
apparent rate constants and amplitudes of 49 (± 1)
s 1 and 74.0 (± 0.1) µM and
0.20 (± 0.03) s
1 and 71 (± 1)
µM, respectively, and with a steady-state release of
phosphate at a rate of 6 µM
1
ArsA.
r). Equilibration of the ArsA
with antimonite leads to further activation of the site that turns over
slowly, so that MgATP turnover is no longer rate-limited by a step
subsequent to hydrolysis. Indeed, the slow burst phase is replaced by
an activity lag, which correlates with the time taken for the
transition between the conformations stabilized by MgATP and Sb(III),
respectively (27). A simple mechanism for Sb(III) to activate the
ATPase would be for antimonite to cause an increase in
k
r, so that the ArsA*-MgADP·Pi intermediate does not build up. In the present investigation we have
provided evidence that Sb(III) destabilizes an
ArsA-MgADP·Pi complex, possibly
ArsA*-MgADP·Pi.
View larger version (5K):
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Scheme 4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1, but the MANT-ADP used to displace ADP
might only do so at a single site, the A2 NBS. Furthermore, a 20-fold
lower concentration of MgMANT-ADP can replace bound MgADP on ArsA,
indicating that MANT-ADP is bound with higher affinity than MgADP. On
the other hand, whereas the ArsA-MgMANT-ADP complex readily dissociates upon dilution, the ArsA-MgADP complex does not, and EDTA is required to
dissociate the ArsA-MgADP complex, indicating that MgADP is bound more
tightly than MgMANT-ADP. Furthermore, EDTA-induced dissociation of the
ArsA-MgADP complex occurs at a much slower rate than
koff for MANT-ADP displacement (e.g.
6.6 s
1 versus >223
s
1). A simple and reasonable explanation of
this anomalous behavior is that MANT-ADP displaces ADP from the A2 NBS,
whereas EDTA induces MgADP dissociation from the A1 NBS. Consistent
with this interpretation, an additional fast phase is observed when
MgADP is used to displace Mg·MANT-ADP from ArsA. These experiments
are also consistent with earlier UV-activated nucleotide cross-linking
studies that suggested that the A1 NBS is a high affinity, poorly
exchangeable site, whereas the A2 NBS is a low affinity, easily
exchangeable site (24). An interesting proposal is that the binding of
nucleotides to the A1 and A2 sites is mutually exclusive, with, for
example, the binding of nucleotide to the A1 site transiently locking
the ArsA in an A1 closed/A2 open (e.g. ArsA1) conformation.
Ser and Cys172
Ser
proteins exhibited a 20-fold increase in the concentration of
antimonite required for half-maximal activation, whereas the Cys422-Ser protein exhibited a 200-fold increase
(33). The Pi burst experiments reported herein indicate
that Sb(III) has three major effects as follows: stimulation of the A1
and A2 NBS, so that they under go a burst in Pi production
during a multiple turnover event, followed by further activation of the
A2 site, so that product dissociation is no longer rate-limiting. An
interesting proposal is that the binding of successive antimonite
molecules, which progressively increase the A1-A2 interactions,
controls these events. We note that the high, intermediate, and low
affinity ArsA-MgATP complexes are formed at rates that differ by more
than an order of magnitude between each complex, so that they are
formed successively in time. Only formation of the high affinity
complex occurs at a rate that could control the fast burst phase.
The rate of formation of the intermediate affinity complex, but
not the low affinity complex, is sufficiently fast to control the slow
burst, and formation of the low affinity complex the activation of the
low affinity NBS. However, no slow burst occurs when ArsA is
equilibrated with Sb(III) prior to mixing with MgATP, suggesting that
the binding of the second antimonite may control activation of the low
affinity NBS.
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ACKNOWLEDGEMENT |
---|
The stopped-flow instrument was purchased with grants from the University of Glasgow and the Royal Society.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant GM55425 (to B. P. R.), a Wellcome Research Travel Grant from the Burroughs Wellcome Fund, and an award from the NATO Collaborative Research Grant Program, and grants from the BBSRC and Wellcome Trust (to A. R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence and requests for reprints should be addressed. Tel.: 44-141-330-3750; Fax: 44-141-330-3751; E-mail: A.Walmsley@bio.gla.ac.uk.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008105200
2 This experiment was repeated for several lower concentrations of ADP with the same result that MgADP dissociation is rapid compared with the rate of binding of MANT-ATP to ArsA.
3 A signal of comparable amplitude and rate was obtained when 100 mM EDTA was used to induce dissociation of the ArsA-MgADP complex (Fig. 7A).
4 Since the data were suggestive of tight-binding, we also fitted it to a quadratic equation for a second-order binding process, but this yielded an identical value for the Kd.
5 A second set of data yielded a half-saturation constant of 170 µM.
6 H. Bhattacharjee and B. P. Rosen, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ABC, ATP-binding cassette; NBD, nucleotide binding domains; MANT, 2'-O-(N-methylanthraniloyl); NBS, nucleotide-binding sites; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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