The metal specificity and selectivity of ZntA from Escherichia coli using the acylphosphate intermediate.

ZntA from Escherichia coli is a P-type ATPase that confers resistance to Pb(II), Zn(II), and Cd(II) in vivo. We had previously shown that purified ZntA shows ATP hydrolysis activity with the metal ions Pb(II), Zn(II), and Cd(II). In this study, we utilized the acylphosphate formation activity of ZntA to further investigate the substrate specificity of ZntA. The site of phosphorylation was Asp-436, as expected from sequence alignments. We show that in addition to Pb(II), Zn(II), and Cd(II), ZntA is active with Ni(II), Co(II), and Cu(II), but not with Cu(I) and Ag(I). Thus, ZntA is specific for a broad range of divalent soft metal ions. The activities with Ni(II), Co(II), and Cu(II) are extremely low; the activities with these non-physiological substrates are 10-20-fold lower compared with the values obtained with Pb(II), Zn(II), and Cd(II). Similar results were obtained with DeltaN-ZntA, a ZntA derivative lacking the amino-terminal metal binding domain. By characterizing the acylphosphate formation reaction in ZntA in detail, we show that a step prior to enzyme phosphorylation, most likely the metal ion binding step, is the slow step in the reaction mechanism in ZntA. The low activities with Ni(II), Co(II), and Cu(II) are because of a further decrease in the rate of binding of these metal ions. Thus, metal ion selectivity in ZntA and possibly other P1-type ATPases is based on the charge and the ligand preference of particular metal ions but not on their size.

P-type ATPases are transporters that utilize the energy liberated in the exergonic ATP hydrolysis reaction to translocate positively charged substrates across membranes (1). Their name derives from the fact that they form an acylphosphate intermediate during the reaction cycle, in which the ␥-phosphate of ATP is transferred to a conserved aspartate residue. Thus, P-type ATPases are evolutionarily distinct from other families of ATP-dependent pumps; mechanistically, they belong to the haloacid dehalogenase protein superfamily (2). Ptype ATPases have been divided into five subgroups; one such subgroup, the P1-type ATPases found in archaea, bacteria, and eukarya, transport soft metal cations such as Cu(I), Ag(I), Zn(II), Cd(II), Pb(II), and Co(II) (3)(4)(5)(6). The physiological functions of these pumps include maintaining the homeostasis of essential metals such as Cu(I), Co(II), and Zn(II), as well as mediating resistance to toxic concentrations of Pb(II), Cd(II), Cu(I), and Ag(I). These pumps may also be involved in delivering essential trace metal ions to target enzymes.
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 (7)(8)(9). 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 (10 -17). 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 (18 -20). 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 1-6 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 nonradioactive 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 aminoterminal 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.

EXPERIMENTAL PROCEDURES
Construction of the D436N Mutant-The D436N mutation in wtZntA was generated using the Quickchange site-directed mutagenesis kit (Stratagene). The oligonucleotides (from Integrated DNA Technologies, Coralville, IA) used for constructing the mutant had the sequences 5Ј-CT-CAG-GTG-GCG-TTT-AAT-AAA-ACG-GGT-ACG-CTG-ACC-3Ј and 5Ј-GGT-CAG-CGT-ACC-CGT-TTT-ATT-AAA-CGC-CAC-CTG-AG-3Ј; the altered bases are indicated in boldface. In addition to the desired mutation, a silent base change (underlined base in the oligonucleotides) was used to create a PinA1 restriction site. This was used to rapidly identify the presence of the desired mutation. The entire sequence of the mutant gene was then verified by automated DNA sequencing (DNA Sequencing Core Facility, Wayne State University). The pBAD/ myc-His C expression vector was used to express the D436N mutant protein in strain LMG194 (zntA::kan), as previously described for ZntA (22). The expression level of the mutant, checked by Western blotting using anti-His tag antibody, was similar to that for wtZntA.
Acylphosphate Formation in Membrane Vesicles with [␥-32 P]ATP-Reactions were carried out with membranes containing ϳ50 g of total protein in 100 l of 20 mM BisTris propane, pH 6.0, containing 200 mM KCl and 10 M EDTA. Following incubation on ice for 5 min, metal-salt solution was added to the membranes at a final concentration of ϳ40 M and incubation continued for another 10 min. The reaction was then started with the addition of 5 mM MgCl 2 and 1 M [␥-32 P]ATP (PerkinElmer Life Sciences). After the desired time interval, the reaction was stopped with 40 l of 10% ice-cold trichloroacetic acid and 1 mM NaH 2 PO 4 . 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 NaH 2 PO 4 before resuspension in acidic buffer (25 mM H 3 PO 4 , pH 2.4, 5% SDS) at room temperature for 3-4 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 Cyclone TM 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 M 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 Proteins-wtZntA and ⌬N-ZntA were purified as described before (24). Phosphorylation of the purified proteins with [␥-32 P]ATP was carried out in 100 l of reaction mixture containing 50 mM Tris-Cl, pH 7.0, 0.1% asolectin, 10% glycerol, 4 g of pure protein pretreated with 2 mM dithiothreitol, and 50 M appropriate metal-salt solution. The reaction mixture was incubated at room temperature for 10 min and the reaction initiated with 10 l of 100 mM MgCl 2 and 2.5 Ci of [␥-32 P]ATP (final concentration, 1 M) at room temperature or 37°C. After 20 s, the reaction was stopped with 500 l of 10% ice-cold trichloroacetic acid and 1 mM NaH 2 PO 4 . 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 NaH 2 PO 4 followed by resuspension in acidic buffer (25 mM H 3 PO 4 , pH 2.4, 5% SDS). Blank samples were obtained by adding stop solution before the addition of [␥-32 P]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 M 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 NaH 2 PO 4 , centrifuged, and worked up as described earlier.
Acylphosphate Formation with [ 32 P]P i -Purified ZntA (4 g) pretreated with 2 mM dithiothreitol was incubated in 100 l of 20 mM BisTris propane, pH 6.0, containing 200 mM KCl, 10 M EDTA, and 0.1% asolectin at room temperature for 5 min, followed by the addition of either 3 l of H 2 O or 1 mM metal salt solution. Following a further incubation for 5 min, the reaction mixture was initiated with 10 l of 100 mM MgCl 2 (final concentration, 9 mM) and 2 Ci of [ 32 P]P i (final concentration, 20 M). After 2 min, the reaction was stopped with 500 l of 10% cold trichloroacetic acid containing 1 mM NaH 2 PO 4 , 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 NaH 2 PO 4 and resuspended in 40 l of 25 mM H 3 PO 4 , pH 2.4, containing 5% SDS for 1 h at room temperature. Aliquots were directly counted in a scintillation counter.
Vanadate Inhibition of ZntA-Inhibition 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 Na 3 VO 4 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 g of purified protein with different concentrations of vanadate together with 2 mM MgCl 2 and 50 M lead acetate for 10 min at 37°C prior to initiating the reaction with 1 M [␥-32 P]ATP and MgCl 2 (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.

Metal Specificity of Acylphosphate Formation in Membrane
Vesicles-As expected for a P-type ATPase, membrane vesicles expressing recombinant ZntA were able to form the acylphosphate intermediate in the presence of ATP and the metal ions Pb(II), Cd(II), and Zn(II) that were previously shown to be the substrates of this transporter (Fig. 1A). Surprisingly, when other metal salts were tested, we observed that ZntA was able to form the intermediate in the presence of Co(II), Cu(II), and Ni(II), divalent metal ions not previously identified as substrates of ZntA (Fig. 1A) (Fig. 1B). It is to be noted that different concentrations of phosphorylated protein were loaded in Fig. 1A such that the total radioactivity in each lane is the same; in Fig. 1B, the same concentration of protein is loaded in each lane and illustrates the different levels of acylphosphate formed for the different metals.
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 Membranes-We 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.
Steady-state Levels of Acylphosphate Intermediate for ZntA with Different Metals-We 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 10 -20 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.
Acylphosphate Formation with Purified Proteins-We next characterized the intermediate formed with purified protein in  (data not shown). In contrast, the K m obtained previ-ously for the steady-state ATP hydrolysis activity of purified ZntA was 106 Ϯ 13 M 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 IC 50 of ϳ1.5 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 (28 -30). However, the IC 50 obtained for the inhibition of the intermediate formation by vanadate for purified ZntA was ϳ50 M (Fig.  4A). This value is similar to the IC 50 of Ͻ20 M, 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.
Kinetics of Dephosphorylation of the Acylphosphate Intermediate with EDTA and ADP-The 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 E 2 P 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 E 1 P and E 2 P 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 E 1 state for Cd(II) relative to Pb(II) and Zn(II), and the E 1 P to E 2 P conformational change in ZntA may be partially ratelimiting with Cd(II) as the substrate.
Acylphosphate Formation with [ 32 P]P i -P-type ATPases can form the acylphosphate intermediate with inorganic phosphate via phosphorylation of the E 2 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 E 1 conformational state, is expected to favor the equilibrium toward the E 1 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 ϳ1.5 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 E 1 state is slow relative to the rate of phosphorylation of the E 2 state such that the presence of metals does not result in a significant fraction of the transporter being present in the E 1 state. DISCUSSION Despite highly homologous sequences, P1-type ATPases that have been characterized to date have been shown to be specific for either Cu(I) and Ag(I), or for Pb(II), Zn(II), and Cd(II). ZntA is the best characterized divalent metal ion-dependent P1-type ATPase to date. We have previously shown that ZntA displays in vivo resistance and in vitro ATPase activity with Zn(II), Cd(II), and Pb(II) (18,21). Unlike Cu(I) and Ag(I), substrates of the monovalent metal ion P1-type ATPases, Pb(II), Zn(II), and Cd(II) do not belong to the same group in the periodic table and have substantially different properties. Our long term goal is to understand the molecular basis of metal ion specificity and selectivity displayed by these pumps. One of our goals in this study was to determine whether the metal specificity of ZntA and its homologues is confined strictly to the three metal ions observed previously or whether other divalent metal ions are also recognized by ZntA, albeit with lower selectivity. We also wanted to reexamine whether Ag(I) is a substrate for ZntA, as had been reported earlier (23). Given the low catalytic activities of P1-type ATPases that have been characterized until now, it is technically challenging to identify additional metal ion substrates that may have extremely low activities, especially using ATP hydrolytic assays. This is, in part, because of the high levels of background ATPase activities resulting from the presence of other types of ATPases typically present in membranes. 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 F 1 F 0 -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 (k 1 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 E 2 state. When ADP is present, E 1 P, but not E 2 P, can transfer the phosphate group to ADP; thus the intermediate decays from the E 1 P 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 E 2 P pathway, once again suggesting that for the reaction with Cd(II), a second step, possibly the E 1 P-E 2 P transition is partly ratelimiting. When the acylphosphate was formed with inorganic phosphate for purified ZntA and ⌬N-ZntA, the presence of metal ions, which bind to the E 1 state and push the equilibrium to the E 1 state, did not affect the level of the intermediate. These results also support our conclusion that the rate of metal ion binding to the E 1 state is slow relative to the rate of phosphorylation of the E 2 state.
Interestingly, the level of acylphosphate intermediate accumulated for ⌬N-ZntA was ϳ2-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 E 1 P-E 2 P transition or metal ion release from E 2 P (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 K m 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 K m 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 K m for ATP obtained with ZntA for the acylphosphate intermediate activity is ϳ2.6 M. Similar values have been obtained for CopA from Archaeoglobus fulgidus and the Wilson's disease Cu(I)-ATPase (16,31). In contrast, the K m for ATP obtained with ZntA for the ATP hydrolysis activity is ϳ106 Ϯ 13 M at pH 7.0 and 37°C (27); a value of 250 M was obtained for CopA from A. fulgidus (16). This difference in K m 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 Ϯ 0.02 nmol/mg of protein at room temperature in the presence of Pb(II). This value is ϳ5-fold lower than that obtained with CopA from A. fulgidus but similar to the plant plasma membrane H ϩ -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. CONCLUSION In this study we show that ZntA from E. coli shows activity specifically with divalent soft metal ions; the monovalent ions, Cu(I) and Ag(I), are not substrates. Specificity toward divalent metal ions is broader than we previously suspected. Using the sensitive acylphosphate formation activity, we show that in addition to Pb(II), Zn(II), and Cd(II), three other metal ions, Ni(II), Co(II), and Cu(II), are also substrates of this pump, though with considerably lower activity. ⌬N-ZntA, a ZntA mutant lacking the amino-terminal metal-binding domain, displays the same metal specificity and selectivity as ZntA. We provide evidence that the low activity with the latter three ions is most likely the result of decreased rates of metal ion binding. Our results also lend further support to our earlier hypothesis that metal ion binding steps are primarily rate-limiting for ZntA and perhaps other P1-type ATPases. Soft-metal ions are believed to bind to ZntA primarily through cysteine ligands (33). Thus, ZntA appears to discriminate between metal ions based on charge and also on the affinity of the particular metal ion for sulfur-containing ligands. However, in contrast to P2type ATPases, the metal ion size appears not to be important in determining selectivity because Pb(II) and Cd(II) are much larger than Zn(II), Ni(II), Co(II), and Cu(II); the latter four have similar sizes and charge but display markedly different activities.