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J Biol Chem, Vol. 274, Issue 36, 25849-25854, September 3, 1999
From the Department of Biology, Georgia State University,
Atlanta, Georgia 30303
ArsA, an anion-stimulated ATPase, consists of two
nucleotide binding domains, A1 in the N terminus and A2 in the C
terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding
ATP. ArsA is known to form a UV-activated adduct with [ ArsA and ArsB proteins together form a transport complex for
efflux of arsenite or antimonite in the inner membrane of
Escherichia coli. ArsA is a peripheral membrane protein that
forms the catalytic component of this transport complex (1, 2). It is
of interest to determine how the energy of ATP hydrolysis by ArsA is
transduced to the integral membrane protein ArsB that forms the carrier
for the anions through the membrane. To obtain insights into how ArsA and ArsB proteins interact to form this primary pump, it is important to understand the mechanism of function of ArsA.
ArsA is an anion-stimulated ATPase that consists of two consensus
nucleotide binding sites, one in the N-terminal (A1) and other in the
C-terminal (A2) half of the protein (1). The A1 and A2 domains of ArsA
show significant internal sequence homology. An alignment of their
primary amino acid sequence suggests that these two domains are
connected by a linker region of about 40 amino acid residues (residues
283-323), the significance of which is not clear (1). Mutations in
either nucleotide binding fold have been shown to result in a loss of
the anion-stimulated ATPase activity (3, 4), implying that both sites
are required for function of the protein. It is not understood how the
A1 and the A2 sites participate in catalysis or whether both sites are
catalytic in nature. ATP binding studies carried out between ArsA and
[ Purification of the ArsA Protein--
Wild type or mutant
His-tagged ArsA proteins were purified from E. coli cells
containing the plasmid pET16barsA as described earlier
(7).
Photolabeling of the ArsA Protein with [32P]ATP or
[3H]ADP--
Purified wild type and mutant ArsA proteins
were photolabeled in the presence of [32P]ATP (3000 Ci/mmol) or [3H]ADP (25 Ci/mmol) by a modification of the
procedure described earlier (5, 8). Each reaction contained 10 µCi of
labeled ATP or ADP. The samples were preincubated in the presence or
absence of antimonite or magnesium for 10 min at 37 °C as indicated
in the figure legends. Photolabeling reactions were carried out on ice
for 30 min with a short wavelength (254 nm) hand-held torch. The
photolabeled samples were analyzed by SDS-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel, followed by
autoradiography. Where indicated, the reactions were quenched by the
addition of excess ADP or ATP to the sample before exposure to UV light.
Digestion with V-8 Staphylococcal Protease--
The labeled
proteins from above were precipitated with 4 volumes of 10%
trichloroacetic acid for 30 min on ice. The precipitated protein was
washed three times with ether, air dried, and dissolved in 0.1 ml of 4 M urea, pH 8.0. V-8 protease was added at a 10:1 ratio of
protein to protease, and the samples were incubated at 37 °C for
16 h. The samples were analyzed by a 12% polyacrylamide gel using
the Tricine1-SDS
electrophoresis system (9).
ATPase Activity of ArsA--
ATPase activity of ArsA was
determined by measurement of inorganic phosphate released from
[ UV Cross-linking of ArsA with [32P]ATP
ArsA has previously been shown to form an adduct with
[ Role of Antimonite--
To determine the effect of antimonite on
UV cross-linking of ArsA with [ Labeling with [ Labeling with [3H]ADP--
To verify that it is
indeed ADP that forms the adduct with ArsA, [3H]ADP was
used directly in the cross-linking reaction. Data in Fig.
2 show that ArsA can be cross-linked to
ADP on exposure to UV light (lane 1). The presence or
absence of antimonite or magnesium did not affect the adduct formation
significantly (lanes 1-4). Presence of excess cold ADP in
the reaction quenched the reaction completely (lane 5). From
the data shown in Figs. 1 and 2, it can be concluded that the adduct on
exposure to UV light is between ArsA and ADP and that increased
hydrolysis of ATP by ArsA in the presence of antimonite results in
increased adduct with ADP.
Labeling of the A1 or A2 Mutants with
[ Labeling of the A1 Peptide with
[
The UV cross-linking experiments described above suggest that adduct in
the absence of antimonite results primarily from the function of A1
site, whereas increased adduct on addition of antimonite is the result
of involvement of both A1 and A2. Because the adduct is between ArsA
and ADP, these data suggest that ArsA carries out unisite catalysis
from the A1 site in the absence of antimonite and multisite catalysis
from both A1 and A2 in the presence of antimonite. This is further
supported by the observation that mutants in the A2 site are still able
to form the adduct due to catalysis by A1 and the adduct in these
mutants is not affected by antimonite, whereas mutants in the A1 site
do not form the adduct due to absence of both unisite and multisite catalysis.
Site of the UV-induced ATP Adduct in the Presence or Absence of
Antimonite
The site where the adduct forms in the absence of antimonite has
previously been identified to lie in a 40-amino acid stretch in the
linker region (residues 283-320) connecting the A1 and the A2 domains
(6) (see also Fig. 4, lane 4).
Because enhanced adduct formation was seen under conditions where
antimonite was present, it was of interest to determine whether this
was due to the formation of the adduct at an additional site, perhaps resulting from catalysis in the A2 domain. In the present study, the
same amounts of the the ArsA protein labeled with
[ Unisite and Multisite Hydrolysis of [ To further analyze unisite and multisite catalysis by ArsA, the
amounts of phosphate released upon hydrolysis of ATP from A1 or from A1
and A2 together were determined. [
The Anion-stimulated ATPase ArsA Shows Unisite and Multisite
Catalytic Activity*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP in the linker region. This study shows
that on addition of antimonite, much more adduct is formed.
Characterization of the nature of the adduct suggests that it is
between ArsA and ADP, instead of ATP, indicating that the adduct
formation reflects hydrolysis of ATP. The present study also
demonstrates that the A1 domain is capable of carrying out unisite
catalysis in the absence of antimonite. On addition of antimonite,
multisite catalysis involving both A1 and A2 sites occurs, resulting in
a 40-fold increase in ATPase activity. Studies with mutant proteins
suggest that the A2 site may be second in the sequence of events, so
that its role in catalysis is dependent on a functional A1 site. It is
also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite
and multisite catalytic conditions, may play an important role in the
energy transduction process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP by UV cross-linking showed that only the A1
domain is involved in forming a UV-activated adduct in the presence of
ATP (4, 5). The site of the adduct was identified to lie in the linker region connecting the A1 and the A2 domains (6). Mutations in the A1
nucleotide binding fold resulted in an inability of the protein to form
the adduct, whereas mutations in the A2 domain had no effect (3, 4),
indicating that the A1 domain is crucial for formation of the ATP
adduct with ArsA. The reason for lack of a similar adduct with ATP in
the A2 domain has not been clear. That the A2 half of the protein
actually binds nucleotide was recently shown by use of an ATP analogue,
5'-p-fluorosulfonylbenzoyladenosine, which was shown to bind
preferentially to the A2 site (7). In that study, it was suggested that
the A1 and A2 sites of ArsA have different conformations and different
affinities for ATP. It was proposed that the A1 site is a high affinity
ATP site that binds ATP in the absence of the ligand, whereas the A2
site has a very low affinity, and the binding of the ligand, arsenite
or antimonite, acts as a switch that allows ATP binding to the A2 site.
However, it is not clear whether two sites in ArsA have independent
catalytic activity or what is the mechanism for catalytic co-operativity in this protein. The present study shows that the A1
domain of ArsA is capable of carrying out unisite catalysis in the
absence of antimonite at a low rate. When the A2 site is also occupied
with ATP, in the presence of antimonite, the catalytic activity
increases. The data shown here also suggest that the A2 site is not an
independent site and that it comes into action only after the A1 site
is occupied, indicating antimonite-induced interaction between the A1
and A2 sites and thus providing first biochemical evidence of
co-operativity between the the two sites in ArsA.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP. Activity was measured in a 40-µl reaction
volume containing 50 mM MOPS-KOH, pH 7.5, 1 mM
dithiothreitol and the indicated amounts of the ArsA protein and
[-32P]ATP (3000 Ci/mmol). Where indicated, the samples
were preincubated with 0.5 mM antimonite at 37 °C for 10 min. The reaction was started by addition of 5 mM magnesium
chloride and the incubation was continued at 37 °C. At the indicated
time points, 5-µl samples were withdrawn, and the reactions were
stopped by adding 2 µl of 10% SDS solution. The reactants and the
products were separated by spotting 2 or 3 µl of the reaction on a
polyethyleneimine TLC plate. Chromatography was carried out with a
mobile phase consisting of 1 M formic acid and 0.7 M LiCl (10). The radioactivity in the resolved spots was
quantitated with a Fuji phosphoimager, and the amount of
32Pi released was calculated in nmol/mg of the
ArsA protein. To determine kinetic parameters of hydrolysis for unisite
or multisite catalysis, ATPase activity at different concentrations of
ATP was determined in the absence or presence of antimonite. The
specific activity of [-32P]ATP was kept constant at
all the concentrations of ATP tested. The data were analyzed by the
enzyme analysis software Enzfitter using the Michaelis-Menten kinetics equation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP on activation with UV light (6, 11). In the
present study, effect of antimonite on the cross-linking reaction was
investigated. These experiments led to a reevaluation of the nature of
the adduct and have provided significant new insights into the
mechanism of function of the enzyme.
-32P]ATP, one set of
samples was preinucbated in the presence of antimonite at 37 °C
before exposure to UV light. Data shown in Fig.
1A indicate that the sample
that had been preincubated with antimonite contained much higher levels
of the adduct (lane 3) compared with the sample that either
contained no antimonite (lane 2) or the sample that
contained antimonite but was not preincubated at 37 °C (lane
1). Because antimonite is known to bring about stimulation of
hydrolysis of ATP by ArsA (2), increased labeling in the presence of
antimonite may be resulting from increased hydrolysis of ATP by ArsA
rather than due to enhanced binding of ATP. Addition of excess cold ADP
or ATP to the reaction just before exposure to UV light was found to
quench the adduct. At 0.5 and 1.0 mM concentrations of the
cold nucleotide, ADP was found to be more effective at quenching the
reaction compared with ATP as seen in Fig. 1B, lanes 1-4,
but at 5.0 mM, both quenched the reaction completely
(lanes 5 and 6).
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Fig. 1.
Characterization of the UV-activated adduct
between ArsA and [32P]ATP. Light-activated adduct
formation between ArsA (final concentration, 7.5 µM) and
[ -32P]ATP (10 µM) or
[-32P]ATP (10 µM) was performed as
described under "Materials and Methods." Samples were preincubated
at 37 °C in the presence or absence of magnesium (final
concentration, 5 mM) and in either the presence or the
absence of antimonite (final concentration, 20 µM) as
indicated. A, effect of antimonite on adduct formation with
[-32P]ATP. All samples contained magnesium.
Lanes 1 and 3, with antimonite. Lanes
2 and 4, no antimonite. Lanes 1 and
2, no preincubation at 37 °C. Lanes 3 and
4, samples were preincubated at 37 °C for 10 min before
cross-linking. B, effect of excess cold ADP or ATP on adduct
formation with [-32P]ATP. Preincubations were carried
out as indicated. The indicated concentrations of cold ADP or ATP were
then added, and samples were exposed to UV light as described below.
Lanes 1 and 2, no addition. Lanes 3 and 4, 0.5 mM. Lanes 5 and
6, 1.0 mM. Lanes 7 and 8,
5.0 mM. Lanes 3, 5 and 7, ADP.
Lanes 4, 6, and 8, ATP C, comparison
of UV-activated adduct formation between ArsA and
[-32P]ATP or [-32P]ATP. The reactions
contained either [-32P]ATP or
[-32P]ATP. Lanes 1, 3, 5, and 7, no antimonite. Lanes 2, 4, 6, and 8, with
antimonite. Lanes 3, 4, 7, and 8, preincubated at
37 °C for 10 min. Lanes 1, 2, 5, and 6, not
preincubated. Following the preincubation in A, B, or C, the samples
were transferred to ice and subjected to UV cross-linking for 30 min.
The samples were analyzed by 10% polyacrylamide-SDS gel
electrophoresis, followed by autoradiography. About 20 µg of protein
was loaded in each lane.
-32P]ATP--
To determine the
nature of the adduct in the UV cross-linking reaction, labeling of ArsA
in the presence of [-32P]ATP and
[-32P]ATP was compared. Data in Fig. 1C
indicate that much less adduct is formed in the presence of
[-32P]ATP (lanes 5-8) compared with when
[-32P]ATP is used (lanes 1-4), indicating
that the adduct might be formed between ArsA and [32P]ADP
resulting from hydrolysis of [-32P]ATP.
[32P]ADP would not result from hydrolysis of
[-32P]ATP, thus explaining the absence of a detectable adduct.
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Fig. 2.
UV-activated adduct between ArsA and
[3H]ADP. Light-activated adduct formation between
ArsA (10 µM) and [3H]ADP (10 µM) was carried out as described under "Materials and
Methods." The reaction mix contained antimonite or magnesium as
indicated. Lane 1, with magnesium. Lane 2, no
magnesium. Lane 3, with antimonite. Lane 4, with
antimonite and magnesium. Lane 5, with 5 mM cold
ADP.
-32P]ATP--
Mutants in the A2 domain of ArsA,
which contain a normal A1 domain, have previously been shown to form an
adduct in the presence of ATP on UV cross-linking (4), whereas mutants
in the A1 domain do not form the adduct (3). Data in Fig.
3A indicate that, unlike the
case with the wild type protein (lanes 1 and 2),
preincubation with antimonite has no significant effect on the adduct
formed with the A2 mutants GA337 or GR337 (lanes 3-6). A1
mutant GS20 showed no adduct in the presence or absence of antimonite
(lanes 7 and 8).
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Fig. 3.
Characterization of the UV-activated adduct
between A1 or A2 mutant proteins or A1 peptide N35 and
[ -32P]ATP. N35 peptide was
expressed in E. coli cells, and the protein was recovered
from the inclusion bodies as described earlier (17). All samples were
preincubated at 37 °C for 10 min. The experimental conditions for
analysis of the light-activated adduct were same as described in the
legend to Fig. 1. A, A1 (G20S) or A2 (G337A or G337R)
mutants. Lanes 1 and 2, wild type. Lanes
3 and 4, GA337. Lanes 5 and 6, GR337. Lanes 7 and 8, GS20 B, N35.
Lanes 1-4, N35. Lanes 5-8, wild type
-32P]ATP--
Previous studies have indicated that
the N-terminal peptide N35 (residues 1-323), consisting of the A1
domain plus the linker, forms an independent domain capable of forming
an adduct in the presence of ATP on exposure to UV light (5). Results
in Fig. 3B indicate that, as in the case of A2 mutants,
antimonite has no effect on the amount of the adduct formed with N35
(lanes 1 and 2), indicating that the A2 domain is
required for the stimulatory effect of antimonite.
-32P]ATP in the absence or presence of antimonite
were subjected to complete digestion with the V-8 protease, as carried
out earlier (6). Data in Fig. 4 suggest that in both samples (labeled
in either the presence (lane 3) or the absence (lane
4) of antimonite), the same 5-kDa fragment is labeled. The 5-kDa
fragment originating from the protein labeled in the presence of
antimonite contained much more adduct (lane 3) compared with
the fragment originating from the protein labeled in the absence of
antimonite (lane 4). These results suggest that the site of
adduct under both conditions is same, and only the amount of the adduct
is different; in either case, the adduct lies in the linker region.
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Fig. 4.
Localization of the UV-activated adduct
between ArsA and [ -32P]ATP.
The ArsA protein UV cross-linked with [-32P]ATP in the
presence or absence of antimonite was subjected to complete V-8
digestion as described under "Materials and Methods." The samples
were analyzed by 12% polyacrylamide Tricine-SDS gel electrophoresis.
Lanes 1 and 3, labeled in the presence of
antimonite. Lanes 2 and 4, labeled in the absence
of antimonite. Lanes 1 and 2, undigested
controls. Lanes 3 and 4, digested with V-8
protease. 25 µg of protein was loaded in each lane.
-32P]ATP by
ArsA
-32P]ATP was
employed in these experiments, and the amount of
32Pi released was determined by TLC as
described under "Materials and Methods." The autoradiogram of a TLC
obtained from a typical experiment is shown in Fig.
5. In this experiment, the wild type ArsA
protein was incubated with [-32P]ATP in the presence
or absence of magnesium and antimonite. The upper spot in
the autoradiogram is the released 32Pi, whereas
the lower spot represents the unhydrolyzed
[-32P]ATP. The results in Fig. 5 show hydrolysis of
ATP in the presence of magnesium without the addition of antimonite
(lane 1) as predicted from UV cross-linking experiments
mentioned earlier (Figs. 1 and 2). On addition of antimonite, the
amount of 32Pi released is significantly higher
(Fig. 5, lane 3). In the absence of magnesium, no ATP
hydrolysis was observed either in the presence (lane 4) or
in the absence (lane 2) of antimonite, which indicates that
the catalysis observed in lane 1 (in the presence of
magnesium and absence of antimonite) is significant and that a two-step catalysis mechanism exists; the first step involves the function of A1
in the presence of magnesium but absence of antimonite, and second step
involves function of both A1 and A2 in the presence of magnesium and
antimonite. Hydrolysis by ArsA in the absence of antimonite has been
observed before (2); however, the fact that it is the result of unisite
catalysis from A1 could not be deduced from earlier observations. In
this study, it was also found that the A2 mutants are able to carry out
unisite catalysis at almost the same rate as the wild type protein;
however, on addition of antimonite, no further increase in the
Pi released was seen, as would be expected due to a
mutation in the A2 site. A1 mutants showed a much lower rate of
32Pi release, implying absence of both unisite
and multisite catalysis (data not shown).
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Fig. 5.
ATPase activity of ArsA by the
32Pi release assay using
[ -32P]ATP. ATPase activity
of the wild type ArsA protein was analyzed as described under
"Materials and Methods." 40 µl reaction contained 50 µg ArsA
protein and 20 µCi [-32P]ATP. Where indicated, the
samples contained 0.25 mM antimonite and/or 5 mM magnesium. The incubation was carried out at
37 °C for 30 min, at the end of which, 6 µg of protein was
spotted on the TLC plate. The TLC plate was developed as described
under "Materials and Methods," followed by autoradiography.
Kinetic Parameters
Because ATP is hydrolyzed by ArsA at different rates under
conditions utilizing the A1 domain alone (in the absence of antimonite) or A1 and A2 domains together (in the presence of antimonite), it was
of interest to determine the Km and
Vmax of the protein under these conditions.
Hence, the initial rate of hydrolysis by ArsA at several different
concentrations of ATP was determined. Fig.
6 shows the rate of
32Pi released at different concentrations of
ATP in the absence and presence of antimonite. The kinetic constants
obtained upon analysis of the data by the Lineweaver-Burk plot suggest
that the Vmax of the A1 site alone is about
th the Vmax obtained for the A1 + A2
sites together. However, Km of the A1 site alone or
A1 + A2 sites together lies within the same range, which is around
150-200 µM, implying that the A1 site by itself has a
high affinity for ATP that is equal to the overall affinity obtained in
the presence of antimonite. Km and Vmax data in the presence of antimonite have
been reported earlier (2), and the values obtained in this study are in
the same range as before.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In most positively regulated allosteric proteins, the binding of an effector serves to increase the affinity of the enzyme for its substrate. Hence, the effect of the ligand can be bypassed by increasing the substrate concentration. However, allosteric regulation of the ATPase activity of ArsA by antimonite appears to occur by a novel mechanism, in which binding of antimonite acts as an "on/off" switch that allows ATP binding to the A2 site (7). The A1 site of ArsA is a high affinity site, and it does not require antimonite for ATP binding; however, the A2 site appears to be inaccessible to ATP in the absence of the ligand (7). This novel mechanism of allosteric regulation in ArsA results from the fact that the two ATP binding sites in ArsA have different conformations, and the A2 site is in an "open" conformation only when the ligand is present. In the present study, it is shown that the A1 site of ArsA is capable of carrying out unisite catalysis in the presence of magnesium alone; however, the addition of antimonite brings about an involvement of the A2 site, thus switching the protein from unisite to multisite catalysis. For full catalytic activity of ArsA, an interaction between A1 and A2 is required which is brought about by antimonite. Whether the A2 site also carries out unisite catalysis is not clear at the moment.
Unisite catalytic activity of the A1 site is strongly supported by UV
cross-linking experiments carried out with [-32P]ATP.
Previous experiments with ArsA resulted in the identification of a
40-amino acid stretch of protein that forms a linker connecting the A1
and the A2 domains as the site where the UV-activated adduct lies (6).
The adduct formation was abolished in mutations in the A1 Walker A
sequence but was unaffected by mutations in the A2 domain (3, 4),
suggesting that only the A1 domain of ArsA is involved in binding ATP
in the UV-catalyzed reaction. The reason for the lack of a similar
adduct in the A2 domain has been elusive. Experiments described in this
paper provide an interesting explanation for the absence of the adduct
in A2: it might lie in the fact that the site identified earlier is
shared between A1 and A2 and that the adduct is the result of stepwise
catalytic reactions, involving first A1 and then A2, as described below.
The data shown here suggest that UV cross-linking actually reflects
binding of ADP and not binding of ATP as assumed earlier. Hence, the
site of adduct formation, identified earlier, is the site where the
product of the reaction ADP binds. Second, it is shown that addition of
antimonite enhances the adduct formation significantly. If the adduct
is between ArsA and ADP, thus reflecting hydrolysis of ATP, then adduct
formation in the absence of antimonite reflects unisite catalysis by
A1, and adduct formation in the presence of antimonite would reflect
multisite catalysis involving A1 and A2. This is supported by the fact
that point mutants in the A1 site do not form the adduct, whereas point
mutants in the A2 site form the adduct. Interestingly, the addition of
antimonite has no significant effect on the adduct formation in A2
mutants, implying the absence of multisite catalysis in these mutants. Hence, A2 mutants seem to carry the first half of the reaction, i.e. unisite catalysis in A1 leading to formation of the ADP
adduct, correctly. These results imply that the A1 site is first in the sequence of events, and A2 comes into play later. Absence of an adduct
in GS20 in either the absence or presence of antimonite suggests that
the A2 site, by itself, is not catalytic/functional, and even though
antimonite brings about the participation of A2 in catalysis, it
requires a functional A1 to do that. Hence, it seems reasonable to
suggest that antimonite brings about an interaction between an intact
A1 and A2 site that may precede ATP binding to A2 resulting in
catalytic co-operativity. It is possible that the A2 site may only play
a regulatory role so that ATP binding to A2 allows much faster product
release from A1 without itself being catalytic in the process. A model
showing unisite and multisite catalysis in ArsA is shown in Fig.
7.
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Unisite and multisite catalytic activity is well documented for the
FoF1 ATPase (12). F1ATPase contains
three catalytic sites on three separate 
subunits. The three sites
in F1 are equivalent; however, they are asymmetric in their affinity
for ATP, resulting in three Km values (13). Which
site is the high affinity site at any particular time is determined by
the rotational position of the subunit with respect to the 33 head. Hence, each site in turn becomes
a high affinity site (14). At substoichiometric concentrations of ATP,
the high affinity site in F1 is filled with ATP and is able
to carry out unisite catalysis. At higher concentrations, ATP binds to
the second and the third sites, and it causes a 106-fold
higher rate of product release from the first high affinity site (13).
ArsA is different in that the asymmetry between the two nucleotide
binding sites in ArsA is present in the structure of the protein. The
A1 site is a high affinity site and is able to carry out unisite
catalysis just like each of the three sites in F1, whereas
binding of ATP to A2 seems to be controlled by a switch, which is the
binding of the ligand antimonite. Filling of the A2 site with ATP
results in catalytic co-operativity between A1 and A2; however, it is
not clear whether, like each of the three catalytic sites in
F1, the A2 site is able to carry out catalysis.
Even though, under multisite catalytic conditions, the amount of the
UV-activated adduct formed with ADP is significantly increased, it was
found that the adduct under both unisite and multisite conditions lies
in the same location, i.e. in the linker region. As
discussed above, UV cross-linking between ArsA and [-32P]ATP identifies a site where the product of the
reaction, ADP, binds. The experiments described here suggest that there
is only one such site in ArsA, thus explaining why it has not been
possible to identify an adduct site specific for the A2 domain. Binding of ADP to the linker under either condition (unisite using A1 or
multisite using A1 and A2) suggests that the linker region might lie at
the interface of the A1 and the A2 domains, and the ADP binding site in
the linker region is a site common to both A1 and A2. In addition, such
a location for binding of ADP might have significant implications in
the energy transduction process. The fact that there is an ADP-bound
conformation resulting from hydrolysis suggests that the ArsA protein
switches between the ATP-bound and the ADP-bound states. It seems
likely that one of these two conformations may be active in terms of
the ability of the protein to interact with the ArsB protein. Hence,
the linker region might be actively involved in the energy transduction
process. Switching between the ADP- and ATP-bound forms of a protein
has been shown for other ATPases, and it has been suggested that this switching is involved in the transduction of energy (15, 16). In the
RecA protein, it has been suggested that, in addition to the Walker A
and Walker B motifs that form the nucleotide binding site, the protein
contains a motif C that undergoes conformational changes upon
hydrolysis of ATP causing it to interact with its substrate DNA (16).
In ArsA, the linker region might be equivalent to the region C of RecA,
and it might be directly involved in interacting with ArsB. This is an
interesting extension of the studies described herein, which needs to
be investigated further. Hence, understanding the biochemical basis of
catalysis in proteins such as ArsA and the conformational changes that
result from catalysis would eventually lead to an understanding of the
mechanism of energy transduction between the catalytic component and
the membrane component of the pump.
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ACKNOWLEDGEMENT |
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I thank P. C. Tai for critical comments and discussions during preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Research Service Award R29 GM51981-02.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 should be addressed: Dept. of
Biology, Georgia State University, 24 Peachtree Center Ave., Atlanta, GA 30303. Tel.: 404-651-3864; Fax: 404-651-2509; E-mail:
bioppk@panther.gsu.edu.
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ABBREVIATIONS |
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The abbreviations used are: Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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): 40-µl reaction volume contained 5 µg of ArsA protein and
different concentrations of ATP at a constant specific activity of
[
): 40-µl reaction volume containing 1.25 µg of ArsA protein and
different concentrations of ATP at a constant specific activity of
[