From the Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201
Received for publication, February 10, 2003 , and in revised form, May 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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P1-type ATPases first became the focus of attention when it was discovered that two human proteins, implicated in Menkes' and Wilson's diseases, were Cu(I) transporters (79). Cu(I) transporters have also been characterized in bacteria and yeast; in addition to Cu(I), some of these pumps have been shown to recognize Ag(I) as a substrate cation (1017). Besides the Cu(I)/Ag(I) transporters, other P1-type ATPases have been characterized that transport soft metal ions such as Pb(II), Cd(II), Zn(II), and Co(II); to date, these divalent soft metal transporters have been found in bacteria and plants (1820). The best characterized transporter from this latter group is ZntA from Escherichia coli, a protein that mediates resistance to toxic concentrations of Pb(II), Cd(II), and Zn(II). Both the Cu(I)/Ag(I) and the Pb(II)/Zn(II)/Cd(II) P1-type ATPases have a distinctive, highly polar metal-binding amino-terminal domain containing 16 repeats of the conserved sequence GXXCXXC, as well as the conserved CPC motif in the sixth transmembrane helix that is believed to be part of the translocation pathway. In fact, all P1-type pumps are highly homologous to each other; hence, the basis of metal ion specificity in these pumps remains an intriguing question.
Before we attempt to understand how substrate specificity is determined in
P1-type ATPases, we need to establish the full spectrum of all the metal ions
that are recognized as substrates by these pumps. We have previously shown
that the physiological substrates for ZntA are Pb(II), Zn(II), and Cd(II)
(18,
21). Also, purified ZntA shows
ATP hydrolysis activity only in the presence of these three metal ions
(22). However, it was reported
that the monovalent ion, Ag(I), induces ATP hydrolysis but not the partial
reaction of acylphosphate formation in ZntA
(23). In this study our goal
was to establish the full substrate spectrum of purified ZntA as well as gain
further insights into the basis of metal ion selectivity in ZntA. We
reexamined the substrate specificity of ZntA using the acylphosphate formation
assay; because this assay has a much lower background activity, it is more
sensitive compared with the ATP hydrolysis assay. Our results clearly showed
that ZntA forms the acylphosphate intermediate not only with the physiological
substrates, Pb(II), Zn(II), and Cd(II), but also the divalent metal ions,
Co(II), Cu(II), and Ni(II). However, no activity was seen with the monovalent
ions Ag(I) and Cu(I). Studies with the D436N mutant, in which the site of
phosphorylation, Asp-436, was altered, showed that this broader metal usage
was indeed because of ZntA and not because of an artifact. Additionally, ATP
hydrolysis activity could be detected with these same divalent metals in
pulse-chase experiments with non-radioactive ATP, though the activity with
these metals is clearly rather low compared with the metal ions that are the
physiological substrates. No ATPase activity was detected with Ag(I) or Cu(I).
The same metal specificity was observed for
N-ZntA,1 a
mutant of ZntA lacking the metal-binding amino-terminal domain
(24). The purified proteins
exhibited a similar substrate profile. Thus, our work clearly shows that ZntA
is strictly specific for divalent metal ions and that the specificity
is broader than we previously believed.
Detailed characterization of the acylphosphate intermediate formed with the six different metal ions with both ATP and inorganic phosphate showed that for ZntA, a step prior to phosphorylation of the enzyme, most likely the metal ion binding step, is rate-limiting. Additionally, the low activity with Co(II), Cu(II), and Ni(II) is because of a slowing down of a step prior to intermediate formation, again, most likely, the metal binding step. Thus, the rate of metal ion binding to ZntA determines the overall rate; metal ion selectivity is a function of how efficiently particular metal ions can bind to ZntA. Our data indicated that ZntA is selective toward divalent soft metal ions that preferentially use sulfur as a ligand. Selectivity in ZntA and possibly other P1-type ATPases is a function of charge and ligand preference of particular metal ions and not because of their overall size.
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EXPERIMENTAL PROCEDURES |
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Preparation of Everted Membrane VesiclesEverted membrane
vesicles were prepared from induced LMG194 (zntA::kan) cells
transformed with plasmids containing ZntA, N-ZntA, or D436N, as
described previously (22).
Membranes were finally resuspended in 25 mM Tris, pH 8.0,
containing 100 mM sucrose, 500 mM NaCl, 1 mM
2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride. The
membrane protein concentration was determined and aliquots were stored at
70 °C until required. For studies with Ag(I), membranes were
centrifuged at 163,000 x g for 90 min. The pellet was then
resuspended in 25 mM Tris, pH 8.0, containing 100 mM
sucrose, 50 mM KNO3, 1 mM 2-mercaptoethanol,
and 1 mM phenylmethylsulfonyl fluoride.
Acylphosphate Formation in Membrane Vesicles with
[-32P]ATP Reactions were carried out
with membranes containing
50 µg of total protein in 100 µl of 20
mM BisTris propane, pH 6.0, containing 200 mM KCl and 10
µM EDTA. Following incubation on ice for 5 min, metal-salt
solution was added to the membranes at a final concentration of
40
µM and incubation continued for another 10 min. The reaction was
then started with the addition of 5 mM MgCl2 and 1
µM [
-32P]ATP (PerkinElmer Life Sciences).
After the desired time interval, the reaction was stopped with 40 µl of 10%
ice-cold trichloroacetic acid and 1 mM
NaH2PO4. The samples were incubated on ice for 10 min
and then centrifuged for 10 min. The pellets were washed four times with 10%
ice-cold trichloroacetic acid and 1 mM
NaH2PO4 before resuspension in acidic buffer (25
mM H3PO4, pH 2.4, 5% SDS) at room temperature
for 34 h. Aliquots were analyzed either on acidic SDS-PAGE or directly
used for scintillation counting (Pharmacia Wallac 1410). Acidic gels were
prepared and run according to established protocols
(25). After electrophoresis,
the gels were dried and analyzed with a CycloneTM storage phosphor system
(Packard).
Pulse-chase experiments of the acylphosphate intermediate were carried out by first forming the intermediate on ice as described above for 20 s, followed by the addition of 100 µM non-radioactive ATP. At fixed intervals the reaction mixture was quenched with trichloroacetic acid. For the zero time point, non-radioactive ATP and trichloroacetic acid were added at the same time.
Acylphosphate Formation with Purified ProteinswtZntA and
N-ZntA were purified as described before
(24). Phosphorylation of the
purified proteins with [
-32P]ATP was carried out in 100
µl of reaction mixture containing 50 mM Tris-Cl, pH 7.0, 0.1%
asolectin, 10% glycerol, 4 µg of pure protein pretreated with 2
mM dithiothreitol, and 50 µM appropriate metal-salt
solution. The reaction mixture was incubated at room temperature for 10 min
and the reaction initiated with 10 µl of 100 mM MgCl2
and 2.5 µCi of [
-32P]ATP (final concentration, 1
µM) at room temperature or 37 °C. After 20 s, the reaction
was stopped with 500 µl of 10% ice-cold trichloroacetic acid and 1
mM NaH2PO4. The samples were incubated on ice
for 10 min and then centrifuged for 10 min. The pellet was washed four times
with 10% ice-cold trichloroacetic acid and 1 mM
NaH2PO4 followed by resuspension in acidic buffer (25
mM H3PO4, pH 2.4, 5% SDS). Blank samples were
obtained by adding stop solution before the addition of
[
-32P]ATP. Aliquots were used for scintillation
counting.
To measure the kinetics of dephosphorylation, purified proteins were first phosphorylated for 20 s at room temperature in the presence of 30 µM metal-salt solution and then incubated with either 10 mM EDTA or 10 mM EDTA together with 2 mM ADP on ice. At fixed time intervals, the reactions were stopped with trichloroacetic acid and 1 mM NaH2PO4, centrifuged, and worked up as described earlier.
Acylphosphate Formation with [32P]PiPurified ZntA (4 µg) pretreated with 2 mM dithiothreitol was incubated in 100 µl of 20 mM BisTris propane, pH 6.0, containing 200 mM KCl, 10 µM EDTA, and 0.1% asolectin at room temperature for 5 min, followed by the addition of either 3 µl of H2O or 1 mM metal salt solution. Following a further incubation for 5 min, the reaction mixture was initiated with 10 µl of 100 mM MgCl2 (final concentration, 9 mM) and 2 µCi of [32P]Pi (final concentration, 20 µM). After 2 min, the reaction was stopped with 500 µl of 10% cold trichloroacetic acid containing 1 mM NaH2PO4, incubated on ice for 10 min and centrifuged for 10 min, in a microcentrifuge. The pellet was washed four times with 10% ice-cold trichloroacetic acid containing 1 mM NaH2PO4 and resuspended in 40 µl of 25 mM H3PO4, pH 2.4, containing 5% SDS for 1 h at room temperature. Aliquots were directly counted in a scintillation counter.
Vanadate Inhibition of ZntAInhibition of both the
Pb(II)-stimulated ATP hydrolysis activity and the Pb(II)-stimulated
acylphosphate formation activity of purified ZntA by vanadate was measured
using a 100 mM Na3VO4 stock solution, pH 7.0,
at 37 °C. The ATPase activities of purified ZntA and the mutant were
assayed either by the pyruvate kinase-lactate dehydrogenase coupled assay or
by a colorimetric method that measures phosphate release at fixed time
intervals (26). The
sensitivity of the acylphosphate intermediate to sodium vanadate was measured
by incubating 4 µg of purified protein with different concentrations of
vanadate together with 2 mM MgCl2 and 50
µM lead acetate for 10 min at 37 °C prior to initiating the
reaction with 1 µM [-32P]ATP and
MgCl2 (final concentration, 8 mM). The reaction mixture
was quenched after 10 s with 10% cold trichloroacetic acid, and was
centrifuged, and the pellet resuspended as described earlier. Aliquots were
used for scintillation counting.
Protein concentrations were determined using the bicinchoninic acid reagent with bovine serum albumin as standard.
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RESULTS |
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To eliminate the possibility that the broader substrate specificity was an artifact caused by using crude membrane vesicles, we tested the ability of different metals to stimulate the intermediate formation in membrane vesicles expressing the D436N mutant of ZntA (Fig. 1C). From homology alignments, Asp-436 is predicted to be the invariant residue that is the site of phosphorylation in ZntA. No intermediate could be detected in the presence of any of the metal ions for the D436N mutant. Thus, it is clear that the intermediate formed in the presence of the six divalent metal ions is because of the presence of ZntA in the membranes; the data also confirm that Asp-436 is the site of phosphorylation in ZntA.
It is evident from Fig.
1A that for ZntA, two protein bands with molecular masses
of 160,000 and 80,000 are phosphorylated; these correspond to the monomer and
dimer forms of ZntA. The dimer form appears to be predominant. On the other
hand, the monomer, with a molecular mass of 69,000, is predominantly observed
for N-ZntA. The active form of P-type ATPases is typically a dimer;
therefore, it is likely that both ZntA and
N-ZntA form dimers, because
both are active proteins (24).
The difference in the phosphorylated protein patterns for ZntA and
N-ZntA is likely because of the fact that the ZntA dimer is stronger
than the
N-ZntA dimer and is not fully disrupted under the mildly
denaturing conditions of the acidic SDS-PAGE.
N-ZntA lacks the
amino-terminal Cys-X-X-Cys motif; these cysteines may
contribute to the additional strength of the dimer formed with ZntA.
Metal Specificity of the Overall ATP Hydrolysis Activity of ZntA in the MembranesWe previously showed that purified ZntA shows detectable ATP hydrolysis activity only with Pb(II), Zn(II), and Cd(II); no activity was observed with Co(II), Cu(II), and Ni(II) (24). We examined the metal specificity of the ATPase activity of crude ZntA in membrane vesicles by measuring the phosphate release activity using a discontinuous colorimetric assay (data not shown). Activity was obtained with Pb(II), Zn(II), and Cd(II); however, activity could not be detected with Co(II), Cu(II), and Ni(II) above that of background levels. This raised the possibility that the metal ions Co(II), Cu(II), and Ni(II) were only able to stimulate the partial reaction of acylphosphate formation in ZntA and not total turnover. Because the level of background activity is low for acylphosphate formation in membranes given that there are only four P-type ATPases in E. coli, we next used this more sensitive method to assay for overall turnover in the presence of these metal ions. Pulse-chase experiments were performed where following formation of the acylphosphate intermediate in the presence of different metal ions, its decay was monitored after the addition of non-radioactive ATP (Fig. 2). The results clearly show that the intermediate disappeared rapidly upon addition of non-radioactive ATP for all the metal ions tested. Because the disappearance of the intermediate in the presence of ATP results from enzyme turnover, it is clear that Ni(II), Cu(II), and Co(II) are also substrates of ZntA, though with very low activities that typically cannot be detected above background levels when the hydrolysis activity is directly assayed.
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Steady-state Levels of Acylphosphate Intermediate for ZntA with Different MetalsWe next investigated the basis of metal selectivity in ZntA, that is the reason why the overall activity with Cu(II), Ni(II), and Co(II) appeared to be much less than with Pb(II), Zn(II), and Cd(II). Fig. 3 shows the steady-state levels of the acylphosphate intermediate formed by ZntA in membrane vesicles for all six metal ions. Steady-state levels were reached within 1020 s for all six metal ions (data not shown). Maximal steady-state levels of the phosphorylated enzyme were accumulated for Cd(II) followed by Zn(II), Pb(II), and Cu(II). Phosphorylated enzyme levels for Co(II) and Ni(II) are much lower. The data suggest that the extremely low ATPase activity of ZntA with Cu(II), Ni(II), and Co(II) is not because of decreased rates of the steps in the reaction following acylphosphate formation but rather because of decreased rates of one or more steps prior to acylphosphate formation. In fact, steps following acylphosphate formation are relatively slow for Cd(II), and to a lesser extent for Zn(II), metal ions that are fairly good substrates (Scheme 1). This conclusion is also supported by Fig. 2, which shows that the acylphosphate intermediate formed with Cd(II) and Zn(II) disappears at the slowest rates when excess cold ATP is added.
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Acylphosphate Formation with Purified ProteinsWe next
characterized the intermediate formed with purified protein in greater detail.
Both purified ZntA and N-ZntA, pretreated with dithiothreitol, were
able to form the acylphosphate intermediate in the presence of ATP and the six
different metal salts. Thus, both purified, detergent-soluble protein as well
as native protein in membrane vesicles exhibit activity with a broad range of
divalent metal ions. Steady-state levels of the intermediate in the presence
of different metal salts were determined at room temperature by measuring time
courses of intermediate formation (data not shown). The steady-state level of
the intermediate was 1020-fold lower for Ni(II), Co(II), and Cu(II)
compared with Pb(II), Zn(II), and Cd(II) for both ZntA and
N-ZntA.
Interestingly, in contrast to the case with ZntA in the membranes, the lowest
level of intermediate formed for purified ZntA was in the presence of Cu(II);
this is most likely because of the reduction of most of the Cu(II) to Cu(I) by
dithiothreitol present in the assay mixture.
The dependence of intermediate formation on metal ion concentration was measured for Pb(II), Zn(II), and Cd(II), the three most active substrate metals (data not shown). The Km values for Pb(II), Zn(II), and Cd(II) were 6.4 ± 1.6, 5.4 ± 1.4, and 4.2 ± 1.4 µM, respectively. These values are similar to the Km values obtained previously for the steady-state ATPase activity of purified ZntA, 5.9 ± 0.4, 5.2 ± 0.7, and 3.8 ± 0.8 µM for Pb(II), Zn(II), and Cd(II), respectively (27). Km values for Ni(II), Co(II), and Cu(II) could not be determined accurately because of the extremely low levels of intermediate formation at low metal concentrations. The dependence of intermediate formation for purified ZntA on the concentration of ATP was measured in the presence of Pb(II); the Km for ATP was 2.6 ± 0.8 µM (data not shown). In contrast, the Km obtained previously for the steady-state ATP hydrolysis activity of purified ZntA was 106 ± 13 µM at pH 7.0 and 37 °C (27).
As expected for the acylphosphate intermediate formed during the reaction
of a P-type ATPase, it was sensitive to vanadate, a classic inhibitor of
P-type ATPases. We measured the inhibition of the intermediate formation by
vanadate in both crude membrane preparations as well as with purified protein.
In crude membrane preparations, we obtained an IC50 of 1.5
mM (data not shown). This value is considerably higher than
corresponding values for P2-type ATPases. Similar observations have been
reported for other P1-type ATPases
(2830).
However, the IC50 obtained for the inhibition of the intermediate
formation by vanadate for purified ZntA was
50 µM
(Fig. 4A). This value
is similar to the IC50 of <20 µM, obtained for the
vanadate inhibition of the ATP hydrolysis activity of purified ZntA
(Fig. 4B). Thus,
vanadate appears to be as potent an inhibitor of ZntA as has been observed for
P2-type ATPases; the weaker inhibition observed in membranes is possibly an
artifact of using crude membrane preparations that have other P-type ATPases
in addition to ZntA.
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Kinetics of Dephosphorylation of the Acylphosphate Intermediate with EDTA and ADPThe decay of the acylphosphate intermediate upon addition of ATP indicated that during overall turnover, the steps following intermediate formation were slowest for Cd(II) and Zn(II) but not for Pb(II) or the slow metal substrates, Ni(II), Co(II), and Cu(II). To confirm this result directly, we measured the rates of dephosphorylation of the acylphosphate intermediate formed by ZntA in the presence of EDTA or EDTA and ADP for the three most active metal substrates. As shown in Fig. 5A, the rate of dephosphorylation in the presence of a strong metal chelator alone, representing dephosphorylation from the E2P state (Scheme 1), is much faster for the intermediates formed with Zn(II) and Pb(II) than with Cd(II). However, the rate of dephosphorylation in the presence of ADP and EDTA, representing dephosphorylation from both the E1P and E2P states, is extremely rapid for the intermediates formed with all three metal ions (Fig. 5B). These data suggest that a similar fraction of the intermediate is present in each conformational state for Pb(II) and Zn(II). However, a slightly larger fraction of the intermediate is in the E1 state for Cd(II) relative to Pb(II) and Zn(II), and the E1P to E2P conformational change in ZntA may be partially rate-limiting with Cd(II) as the substrate.
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Acylphosphate Formation with
[32P]PiP-type ATPases can
form the acylphosphate intermediate with inorganic phosphate via
phosphorylation of the E2 state
(Scheme 1). Both purified ZntA
and N-ZntA could be phosphorylated using inorganic phosphate in the
absence of metals. The presence of metal ions, which bind to the
E1 conformational state, is expected to favor the
equilibrium toward the E1 state and hence result in a
lower level of phosphorylation by inorganic phosphate. However, we observed
the presence of metal ions did not have a significant effect on the level of
phosphorylation by inorganic phosphate for either ZntA or for
N-ZntA
(data not shown). This result is in contrast to the conclusion drawn earlier
with ZntA in membranes (23);
however, in that study, results were not quantitated. The maximal amount of
acylphosphate formed with ZntA with inorganic phosphate was
1.5 nmol/mg
of protein at room temperature irrespective of whether metal ions were present
or not. These results demonstrate that the rate of metal ion binding to the
E1 state is slow relative to the rate of phosphorylation
of the E2 state such that the presence of metals does not
result in a significant fraction of the transporter being present in the
E1 state.
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DISCUSSION |
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P-type ATPases differ from F- and V-type ATPases in forming a covalent
intermediate during the reaction cycle in which the -phosphate of ATP
is transferred to a conserved aspartate residue
(1). Because the E.
coli F1F0-ATPase does not form the acylphosphate
intermediate, this is a more sensitive assay to test for additional metal ion
substrates with low activities when the protein is expressed in E.
coli. In this study, using the acylphosphate formation assay, we showed
that recombinant ZntA in membrane vesicles was able to utilize Co(II), Ni(II),
and Cu(II) as substrates in addition to Pb(II), Cd(II), and Zn(II). Thus, the
metal ion specificity is more broadly confined to divalent soft-metal ions
than previously suspected. Our results also unequivocally ruled out monovalent
metal ions such as Ag(I) and Cu(I) as substrates for ZntA. Similar results
were also obtained with
N-ZntA, supporting our earlier conclusion that
the cysteine-rich amino-terminal domain of ZntA is not required for function,
although our data suggest that ZntA forms a stronger dimer when this domain is
present (24). Results with the
D436N mutant also showed that Asp-436 is the site of phosphorylation; the
D436N mutant is completely inactive with respect to both in vivo
resistance and ATP hydrolysis activity.
Though ZntA or N-ZntA was capable of complete turnover in the
presence of Co(II), Ni(II), or Cu(II), the ATPase activities with these metal
ions are extremely low because we could not detect any activity above
background levels when ATP hydrolysis was directly assayed. Consistent with
this observation, we observed that a zntA-deleted strain did not
display increased sensitivity to Ni(II), Co(II), or Cu(II); additionally,
overexpressed ZntA did not confer resistance to toxic concentrations of
Ni(II), Co(II), and Cu(II). Thus, in vivo, ZntA is selective for
Pb(II), Zn(II), and Cd(II) only. Of the three physiological metal ion
substrates, purified ZntA has the highest ATPase activity with Pb(II),
followed by Zn(II) and Cd(II)
(22). A goal of this study was
to understand the basis of metal ion selectivity in ZntA in terms of the
reaction mechanism (Scheme 1).
Steadystate levels of acylphosphate formed with ZntA are highest for Cd(II)
and quite low for the slow substrates, Co(II), Ni(II), and Cu(II). Because an
increased level of accumulation of a reaction intermediate implies that steps
beyond the formation of that intermediate are slow, our results clearly show
that the low activities with Co(II), Ni(II), and Cu(II) are not because of
slow steps following acylphosphate formation but possibly because of slow
steps preceding acylphosphate formation. In general, the steady-state levels
of acylphosphate formation for all six metal ions were quite low, suggesting
that for ZntA, one or more steps preceding acylphosphate formation is
rate-limiting for all the metal ion substrates; it is reasonable to assume
that this is the metal binding step (k1 in
Scheme 1). However, because
comparatively the highest level of intermediate was accumulated for Cd(II), it
is possible that a second step following acylphosphate formation may also
contribute to the overall rate for this particular metal ion.
These conclusions are also supported by our data measuring
dephosphorylation of the acylphosphate intermediate formed with different
metal ions in the presence of either EDTA alone or EDTA and ADP. In the
presence of EDTA alone, the intermediate decays from the
E2 state. When ADP is present, E1P,
but not E2P, can transfer the phosphate group to ADP; thus
the intermediate decays from the E1P state
(Scheme 1). The decay of the
intermediate was quite fast via either pathway, supporting our proposal that
none of the steps involved in the decay of the intermediate is slow. However,
the intermediate formed with Cd(II) decays at a slightly slower rate via the
E2P pathway, once again suggesting that for the reaction
with Cd(II), a second step, possibly the
E1P-E2P transition is partly
rate-limiting. When the acylphosphate was formed with inorganic phosphate for
purified ZntA and N-ZntA, the presence of metal ions, which bind to the
E1 state and push the equilibrium to the
E1 state, did not affect the level of the intermediate.
These results also support our conclusion that the rate of metal ion binding
to the E1 state is slow relative to the rate of
phosphorylation of the E2 state.
Interestingly, the level of acylphosphate intermediate accumulated for
N-ZntA was
2-fold higher relative to ZntA. As noted in a previous
study,
N-ZntA has a slightly lower ATPase activity relative to ZntA
(24). It is possible that a
catalytic step after phosphorylation but prior to dephosphorylation is slower
in
N-ZntA compared with ZntA, for example, the
E1P-E2P transition or metal ion
release from E2P
(Scheme 1). It is not clear,
however, how removal of the amino-terminal metal-binding domain can lead to a
slowing of either of these steps.
The Km values obtained with ZntA for Pb(II),
Zn(II), and Cd(II) for the acylphosphate formation activity are similar to
those we measured previously for the ATPase activity; all three metals have
similar affinities that are in the low micromolar range. These similar
Km values for both the first half-reaction and
overall turnover further support our hypothesis that the slow step in the
reaction occurs before acylphosphate formation. The
Km for ATP obtained with ZntA for the
acylphosphate intermediate activity is 2.6 µM. Similar
values have been obtained for CopA from Archaeoglobus fulgidus and
the Wilson's disease Cu(I)-ATPase
(16,
31). In contrast, the
Km for ATP obtained with ZntA for the ATP
hydrolysis activity is
106 ± 13 µM at pH 7.0 and 37
°C (27); a value of 250
µM was obtained for CopA from A. fulgidus
(16). This difference in
Km values is probably because of the two
different affinities for ATP that have been demonstrated for P-type ATPases
(1). The maximal amount of
acylphosphate formed with ZntA under saturating ATP concentrations is 0.23
± 0.02 nmol/mg of protein at room temperature in the presence of
Pb(II). This value is
5-fold lower than that obtained with CopA from
A. fulgidus but similar to the plant plasma membrane
H+-ATPase (16,
32). These low steady-state
levels of acylphosphate for the P1-type ATPases again suggest that the
rate-limiting steps occur prior to acylphosphate formation for these
transporters.
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CONCLUSION |
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Biochemistry and
Molecular Biology, Wayne State University, School of Medicine, 540 E. Canfield
Ave., Detroit, MI 48201. Tel.: 313-577-0040; Fax: 313-577-2765; E-mail:
bmitra{at}med.wayne.edu.
1 The abbreviations used are: N-ZntA, a mutant of ZntA with residues
2106 deleted; BisTris,
2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)propane-1,3-diol.
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REFERENCES |
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