From the Department of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark
Received for publication, November 29, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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Thr214 of the highly conserved
214TGES sequence in domain A of the
Na+,K+-ATPase was replaced with alanine, and
the mutant was compared functionally with the previously characterized
domain A mutant Gly263 The
Na+,K+-ATPase1
maintains high Na+ and K+ gradients across the
plasma membrane in all animal cells by mediating active transport of
Na+ out and K+ into the cell at a stoichiometry
of 3:2, using the energy derived from hydrolysis of ATP (1). The
reaction steps linking ion transport to ATP hydrolysis through
conformational changes are described by the consecutive
"Post-Albers" or
"E1-E2" model
depicted in Scheme 1 (2-4). The
Na+,K+-ATPase is a member of the family of
P-type ATPases, which include among others the
Ca2+-ATPase and H+,K+-ATPase, and
for which autophosphorylation by ATP of an aspartyl residue is a
characteristic feature of the reaction cycle. In the
Na+,K+-ATPase, the phosphorylation reaction is
triggered by binding of three cytoplasmic Na+ ions to the
E1 form of the enzyme, and dephosphorylation is
activated by the binding of K+ to extracellularly facing
sites. The translocation of Na+ occurs in connection with a
conformational change in the phosphoenzyme, E1P(Na3) Ala.
Thr214
Ala displayed a conspicuous 150-fold reduction
of the apparent vanadate affinity for inhibition of ATPase activity,
which could not simply be explained by the observed shifts of the
conformational equilibria in favor of E1 and
E1P. The intrinsic vanadate affinity of the
E2 form and the effect on the apparent vanadate
affinity of displacement of the
E1-E2 equilibrium were
determined in a phosphorylation assay that allows the enzyme-vanadate
complex to be formed under equilibrium conditions. When the
E2 form prevailed, Thr214
Ala
retained a reduced vanadate affinity relative to wild type, whereas the
affinity of Gly263
Ala became wild type-like. Thus,
mutation of Thr214 affected the intrinsic affinity of
E2 for vanadate. Furthermore, Thr214
Ala showed at least a 5-fold reduced
E2P dephosphorylation rate relative to wild
type in the presence of saturating concentrations of K+ and
Mg2+. Because vanadate is a phosphoryl transition state
analog, it is proposed that defective binding of the phosphoryl
transition state complex (transition state destabilization) causes the
inability to catalyze E2P dephosphorylation
properly. By contrast, the phosphorylation site in the
E1 form was unaffected in Thr214
Ala. Replacement of the glutamate, Glu216, of
214TGES with alanine was incompatible with cell viability,
indicating a very low transport activity or expression level. Our
results support the hypothesis that domain A is isolated in
the E1 form, but contributes to make up the
catalytic site in the E2 and
E2P conformations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E2P transition. The countertransport of
K+ is associated with the dephosphorylation of
E2P and another conformational change,
E2(K2)
E1, which deoccludes K+ at the
cytoplasmic side and is accelerated by binding of ATP (Scheme 1). To
understand the mechanism of cation transport, it is essential to
elucidate the nature of the conformational changes and the catalytic
events, as well as the links between them.
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Scheme 1.
Minimum reaction cycle of the
Na+,K+-ATPase. Occluded ions are shown in
parentheses. Modulatory ATP is shown boxed.
The high-resolution crystal structure of the Ca2+-ATPase in
the E1Ca2 state (5) shows that the
protein is comprised by 10 membrane-spanning segments, M1-M10, and a
cytoplasmic part consisting of three distinct main domains:
A for actuator, N for nucleotide binding, and
P for phosphorylation (i.e. containing the
phosphorylated aspartate residue). The overall structure of the
Na+,K+-ATPase -subunit resembles that of
Ca2+-ATPase (6, 7), and many residues, including those
known to be involved in ion binding and catalysis (8-11), are highly conserved between these two pump proteins. The most conserved sequences2 among P-type pumps
are 371DKTGT (containing the phosphorylated aspartate),
610MVTGD, and 710TGDGVNDS, within domain
P, and 214TGES in domain A (12, 13).
A number of studies have suggested that 214TGES plays an
important role in energy transduction. Proximity relations determined
by specific affinity cleavage have indicated that 214TGES
moves toward domain P during the E1
E2(K2) and
E1P(Na3)
E2P transitions, thereby displacing domain
N from domain P (14). It was hypothesized that
the E1P(Na3)
E2P conformational transition is associated with
a change in Mg2+ ligation, and that the glutamic acid
residue of 214TGES is involved in Mg2+
coordination in E2P, but not in
E1P(Na3) (14). Previous mutagenesis analysis of the Ca2+-ATPase has implicated each of the
residues TGE of the conserved TGES sequence in the
E1P(Ca2)
E2P transition (15). Other residues in domain
A have also been shown by mutagenesis to be involved in the
E2(K2)
E1
and E1P(Na3)
E2P transitions of the
Na+,K+-ATPase. Hence, mutation of
Glu233 in the Na+,K+-ATPase
accelerated the E2(K2)
E1 transition of the dephosphoenzyme (16), and
mutation of Gly263 located in the loop connecting domain
A with M3 (see Fig. 1) interfered with both E2(K2)
E1 and
E1P(Na3)
E2P (17). Similar effects were seen for mutation
of the corresponding residue in the Ca2+-ATPase,
Gly233 (18, 19). Moreover, the results of proteolytic
cleavage studies are consistent with large domain motions in the
cytoplasmic region, involving rotation of domain A, in
connection with the conformational changes (20-23). Very recently, the
crystal structure of the Ca2+-ATPase in the
Ca2+-free E2 state revealed that
large movements of the three cytoplasmic domains occur during the
transition from E1Ca2 to
E2, leading to formation of a compact headpiece
in the E2 state, where the threonine of the TGES
sequence is located at the interface between the A and
P domains close to the phosphorylated aspartate and other
catalytically important residues, see Fig. 1 (24). This could mean that
the threonine plays a role in the catalytic function of
E2 and E2P.
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In the present study, the functional role of Thr214 of the
Na+,K+-ATPase was examined by replacement with
alanine. To analyze both the conformational changes and the function of
the catalytic site in the mutant, we have conducted rapid kinetic
measurements of phosphorylation, as well as dephosphorylation. In
addition, the affinity for the phosphate transition state analog
vanadate was determined both during enzyme turnover and at equilibrium
under conditions where the E2 form prevails. The
results show that Thr214 is important for proper function
of the catalytic site in E2 and
E2P, whereas the catalytic site of the
E1 form seems to function normally in the
Thr214 Ala mutant. The effects of the
Thr214
Ala mutation presented here add functional
evidence to the hypothesis that domain A contributes
substantially to make up the catalytic site in the
E2 and E2P conformations.
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EXPERIMENTAL PROCEDURES |
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Mutagenesis, Expression, and Basic Functional
Characterization--
Oligonucleotide-directed mutagenesis (25) of the
cDNA encoding the ouabain-insensitive rat 1-isoform
of Na+,K+-ATPase, expression of mutants and
wild type in COS-1 cells, using 5 µM ouabain in the
growth medium to select stable transfectants, and the isolation of
crude plasma membranes from the cells were carried out as described
previously (26, 27), and the concentration of total protein was
determined using the dye binding method of Bradford (28).
ATPase activity was measured on sodium deoxycholate- or alamethicin-treated leaky plasma membranes at 37 °C as described previously (26, 27). Titration of the ouabain concentration dependence of the ATPase activity gave similar K0.5 values for the wild type (132 ± 8 µM) and the mutant (95 ± 28 µM). The fraction of active Na+,K+-ATPase molecules in the preparation contributed by the exogenous enzyme calculated as in Ref. 29 was 92% for the wild type and 95% for the mutant, indicating that only a minimal amount of endogenous COS-1 cell enzyme was present. To eliminate the contribution of the latter, 10 µM ouabain was added to all assays. The Na+,K+-ATPase activity associated with the expressed exogenous enzyme was calculated by subtracting the ATPase activity measured at a concentration of ouabain (10 mM) that inhibits all Na+,K+-ATPase activity from that measured at 10 µM ouabain.
Studies of the Na+ dependence of steady-state
phosphorylation from [-32P]ATP and the time course of
ADP-dependent dephosphorylation, and the determination of
the active site concentration by phosphorylation in the presence of 150 mM NaCl and oligomycin (20 µg/ml) to inhibit dephosphorylation were carried out at 0 °C as previously described (30). Deocclusion of K+ was generally studied in
phosphorylation experiments at 10 °C, following formation of
K+-occluded enzyme at room temperature as previously
described (17, 29, 31). To prevent dephosphorylation, oligomycin was
added prior to initiation of phosphorylation with
[
-32P]ATP (31). In addition, some deocclusion
experiments were performed in the same way, but at 0 °C in the
presence of final concentrations of 5 µM
[
-32P]ATP, 5 mM MgCl2, 20 mM Tris (pH 7.5), 1 mM EGTA, 20 µg/ml
oligomycin, and 10 µM ouabain, following incubation at
20 °C for 30 min with 8 mM RbCl, to mimic the conditions
prevailing in the vanadate binding assay described below. In all cases,
background phosphorylation was determined in the presence of 50 mM KCl without NaCl.
Phosphorylation Assay for Determination of Vanadate Binding at
Equilibrium--
To examine the vanadate affinity under equilibrium
conditions, 10 µg of deoxycholate-treated plasma membranes were
incubated at 20 °C for 30 min in 40 µl of medium containing 20 mM Tris (pH 7.5), 5 mM MgCl2, 1 mM EGTA, 10 µM ouabain, and the indicated concentration of orthovanadate. To promote accumulation of the enzyme
in the E2 form, 8 mM RbCl was added
to the medium during the incubation with vanadate. The samples were
then cooled to 0 °C to prevent dissociation of bound vanadate (32),
and the enzyme fraction with no vanadate bound was determined by
measuring the amount of phosphoenzyme formed during a 30-s incubation
following the addition of 360 µl of ice-cold phosphorylation medium
producing final concentrations of 50 mM NaCl, 50 mM choline chloride, 20 mM Tris (pH 7.5), 5 µM [-32P]ATP, 5 mM
MgCl2, 1 mM EGTA, and 20 µg/ml oligomycin.
The data were fitted by assuming a simple one-site binding model, where only the enzyme fraction with no vanadate bound phosphorylates,
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(Eq. 1) |
Rapid Kinetic Phosphorylation and Dephosphorylation
Studies--
To perform rapid kinetic phosphorylation and
dephosphorylation experiments at 25 °C, a Bio-Logic quench-flow
module QFM-5 (Bio-Logic Science Instruments, Claix, France) was used as
previously described (17). The phosphorylation rate of enzyme present
in the Na+-saturated form was determined in single-mixing
experiments carried out as previously described according to
"Protocol 1" (17, 33). To monitor the rate of the
E1P E2P transition or
the dephosphorylation of E2P, a double-mixing
procedure was used to study dephosphorylation of phosphoenzyme formed
either in the presence of 600 mM NaCl to accumulate
E1P ("Protocol 2a") or in the presence of 20 mM NaCl and 130 mM choline chloride to
accumulate E2P ("Protocol 2b") (17, 33).
The time course of dephosphorylation of E1P was
analyzed using the equation describing the kinetics of two consecutive
first-order reactions E1P E2P
E2 with rate
constants k1 and k2,
respectively,
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(Eq. 2) |
Data Analysis and Statistics--
Data normalization, averaging,
and nonlinear regression analysis were carried out as previously (17).
The lines in the figures show the best fit to the complete set of
normalized data with the error bars shown for each data point
corresponding to the standard error. Ligand dependences were analyzed
by applying the Hill equation (17), and the time course of
K+ deocclusion by use of the biexponential equation
described previously (17).
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RESULTS |
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Expression, Na+, K+, and ATP Dependence of
Na+,K+-ATPase Activity, and Catalytic Turnover
Rate--
Thr214 of the rat kidney
Na+,K+-ATPase was replaced with alanine, and
the resulting enzyme was expressed in COS-1 cells under ouabain selective pressure as previously (26, 27). The Thr214 Ala mutant was able to confer ouabain resistance to the cells, indicating that the mutant is able to transport Na+ and
K+ at rates compatible with cell growth. Thr214
Ala was expressed to a level very similar to that of the wild-type enzyme (about 60 pmol/mg of total membrane protein determined as the
maximum phosphoenzyme level, EPmax, see below
and Refs. 30 and 33), allowing the effects of this mutation on the
overall and partial reaction steps of the enzyme cycle to be analyzed. Transfection was also carried out with Glu216
Ala, but
this mutant failed to confer ouabain resistance to the cells,
indicating that the transport activity or the expression level of this
mutant is too low to maintain cell viability.
Titration of the Na+, K+, and ATP dependence of
ATPase activity of Thr214 Ala showed a slightly
increased apparent affinity for Na+ (1.5-fold), a slightly
decreased apparent affinity for K+ (1.2-fold),
and a 3.3-fold increase in apparent
affinity for ATP, relative to wild type (Fig. 2 and Table
I). Because the E1
form binds Na+ and ATP with high affinity, but
K+ with low affinity, and the E2
form binds K+ with high affinity and ATP with low affinity,
the effects of the mutation on the apparent Na+,
K+, and ATP affinities are compatible with a shift of the
E1-E2 conformational
equilibrium of the dephosphoenzyme in favor of E1.
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The maximal catalytic turnover rate of the
Na+,K+-ATPase was estimated as the ratio
between Vmax for ATP hydrolysis and the active
site concentration, determined as the maximum phosphoenzyme level,
EPmax, measured at 0 °C by phosphorylation
with ATP in the presence of 150 mM NaCl and oligomycin to
inhibit dephosphorylation. A value of 3225 ± 262 min1 was found for Thr214
Ala,
i.e. about 38% of the turnover rate of 8474 ± 165 min
1 determined for the wild type (30, 33).
Vanadate Dependence of Na+,K+-ATPase
Activity--
Vanadate is considered an analog of the phosphoryl
transition state and binds specifically to E2
and E2(K2), leading to an inhibited
dead-end state that probably resembles the
E2·P complex from which phosphate is released
(34). Fig. 3 shows titration of the
vanadate inhibition of steady-state
Na+,K+-ATPase activity in the presence of 20 mM K+, i.e. a condition facilitating
vanadate inhibition in the wild type. Relative to the wild type, the
mutant Thr214 Ala displayed a conspicuous 150-fold
reduction of the apparent affinity for vanadate in the ATPase assay.
This suggests either that the steady-state level of the
vanadate-reactive E2 conformation was depleted
or the intrinsic affinity for vanadate reduced in the mutant.
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Na+ Dependence of Phosphorylation--
To reveal the
Na+ affinity at the cytoplasmically facing Na+
sites without interference from K+, the Na+
dependence of phosphorylation from ATP was studied in the absence of
K+ and presence of oligomycin to inhibit dephosphorylation
(Fig. 4). Relative to wild type, the
apparent affinity for Na+ was slightly increased (1.6-fold)
in Thr214 Ala, in line with the above described
increase in the apparent affinity for Na+ in measurement of
Na+,K+-ATPase activity.
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Time Course of Phosphorylation with
[-32P]ATP--
To test the phosphorylation reaction
E1Na3
E1P(Na3) and the ATP affinity of the
E1Na3 form, the time course of
phosphorylation with 2 µM [
-32P]ATP was
studied in the presence of oligomycin (Fig.
5), using a quench-flow module QFM-5 (see
"Experimental Procedures"). The observed rate constant,
kobs, was 32 s
1 for
Thr214
Ala, i.e. very similar to that of the
wild-type enzyme (29 s
1). In the presence of 2 µM ATP, the phosphorylation reaction is less than
half-saturated in the wild type (17), and, accordingly, any difference
between mutant and wild type with respect to the affinity of
E1Na3 for ATP should be reflected in
the kobs value. Hence, the result of the
phosphorylation experiment suggests that in Thr214
Ala
the phosphorylation reaction as well as the binding of ATP to
E1Na3 is wild type-like.
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Occlusion and Deocclusion of K+--
To examine the
amount of K+-occluded enzyme at equilibrium and the rate of
the K+ deocclusion reaction
E2(K2) E1 + 2K+, the previously described phosphorylation assay was
applied (Fig. 6) (17, 29, 31). Following
formation of the K+-occluded complex in the presence of 8 mM K+, the phosphorylation was monitored upon a
10-fold dilution of the enzyme in a solution containing 1 µM [
-32P]ATP and 100 mM
Na+. As previously described for the wild type and other
mutants (17, 29, 31), the time course of phosphorylation can be analyzed as a biphasic time function, in which the component
corresponding to the rapid phase is at maximum from the beginning,
because it reflects the nonoccluded E1 enzyme
pool that binds Na+ and phosphorylates within 5 s. The
slow phase (shown by the lines in Fig. 6) reflects the
phosphorylation of E2(K2) through
the steps E2(K2)
E1
E1Na3
E1P(Na3), where the release of
occluded K+ is rate-limiting. The fitting procedure allows
extraction of the rate constant corresponding to the slow phase and the
amplitude of the slow phase (100% minus the ordinate intercept),
corresponding to the relative amount of enzyme initially present as
E2(K2). These parameters are shown
in Table I. Relative to wild type, the Thr214
Ala
mutant showed a 4-fold increase of the rate of release of occluded
K+, and the relative amount of
E2(K2) was 82% in the mutant
versus 96% in the wild type.
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Vanadate Binding at Equilibrium--
The 150-fold reduction of the
apparent affinity for vanadate determined in the ATPase assay may in
principle arise from a direct effect of the mutation on the
vanadate-binding site (a change to the "intrinsic" affinity for
vanadate) or be secondary to changes in the rates of reactions in the
enzyme cycle preceding and subsequent to the vanadate-reactive
E2 conformation, which would affect the
accumulation of E2 at steady state and, thereby, the sensitivity to inhibition by vanadate. To be able to distinguish between these possibilities, we have adopted a phosphorylation assay
that allows the enzyme-vanadate complex to be formed under equilibrium
conditions, where E2 is not being continuously
produced by dephosphorylation of E2P as during
enzyme cycling. The enzyme is equilibrated with various concentrations
of vanadate at 20 °C in the absence of ATP, and the vanadate-free
enzyme fraction is determined by its ability to form a phosphoenzyme
with [-32P]ATP after cooling to 0 °C. Vanadate
binding is competitive with phosphorylation, and because the
dissociation of vanadate is very slow at 0 °C (32), the amount of
phosphoenzyme formed in this assay reflects the equilibrium between the
free and vanadate-bound enzyme forms existing during the equilibration
with vanadate at 20 °C before the addition of
[
-32P]ATP.
Results obtained with this assay are shown in Fig.
7 and summarized in Table
II. Generally, the vanadate affinity
reported here is higher than that determined during enzyme turnover
with ATP. This is probably because of the lack of competition from ATP
binding and phosphorylation during the equilibration with vanadate.
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Fig. 7A shows results obtained following equilibration with
vanadate in the absence of any of the transported cations. Under these
conditions, Thr214 Ala exhibited 8-fold reduction of
the apparent affinity for vanadate relative to wild type. A change in
this direction could in principle arise from a mutational effect
shifting the E1-E2 conformational equilibrium of the dephosphoenzyme in favor of E1, and, indeed, there is evidence for such a
shift from the changes to the apparent affinities for Na+,
K+, and ATP, as well as the rate of the K+
deocclusion reaction and the equilibrium level of
E2(K2) presented above, although the
changes to these parameters were relatively small (see Table I). For
comparison, Fig. 7 also shows vanadate inhibition results obtained with
the previously characterized mutant Gly263
Ala (17)
that exhibited more pronounced changes than Thr214
Ala
with respect to the parameters characterizing the conformational equilibrium (cf. Table I). As seen in Fig. 1,
Gly263 is located in the loop connecting domain
A with M3. The corresponding glycine in the
Ca2+-ATPase undergoes a large movement in relation to the
E1-E2 transition, but
unlike the threonine does not approach the phosphorylated aspartate. In
accordance with a more pronounced shift of the conformational equilibrium in favor of E1 in Gly263
Ala as compared with Thr214
Ala, the apparent
affinity for vanadate observed in the absence of the transported
cations (Fig. 7A) was also reduced more in Gly263
Ala (22-fold reduction relative to wild type)
than in Thr214
Ala.
An important question was, however, whether the relatively small change
to the E1-E2
conformational equilibrium of Thr214 Ala indicated by
the above mentioned parameters (Table I) is sufficient to account for
the 8-fold reduction in apparent vanadate affinity observed in the
equilibrium binding assay. To eliminate the influence of the shift of
E1-E2 equilibrium
induced by the mutation, the vanadate titration was also performed in the presence of 8 mM Rb+ (Fig. 7B).
As seen in the inset to Fig. 7B, measurements of
occlusion and deocclusion of Rb+, carried out according to
the same principles as for Fig. 6, but under the same conditions as
applied in the vanadate binding assay, showed that in the presence of 8 mM Rb+, the amount of
E2(Rb2) present was close to 100%
for both mutants and the wild type (see legend to Fig. 7). The
main part of Fig. 7B shows that under these
conditions the affinity of Gly263
Ala for vanadate was
wild type-like, as would be expected for a "conformational mutant"
in which the intrinsic affinity of E2 for
vanadate is unaffected by the mutation. By contrast, Thr214
Ala displayed a 4-fold reduced affinity for vanadate relative to
wild type, even under conditions where the enzyme was 100% on the
E2(Rb2) form, indicating that this
mutation affected the intrinsic affinity for vanadate.
ADP Sensitivity of the Phosphoenzyme--
The phosphoenzyme is
usually considered to consist of two major pools,
E1P and E2P, which can be
distinguished by their different reactivities toward ADP and
K+. The pool referred to as E1P is
K+-insensitive but ADP-sensitive, i.e. rapidly
dephosphorylated by ADP because of its ability to donate the phosphoryl
group back to ADP forming ATP, whereas E2P is
ADP-insensitive and K+-sensitive, i.e. rapidly
hydrolyzed in the presence of K+, binding at
extracellularly facing sites. The partition of the phosphoenzyme
between these two pools at steady state can be analyzed by studying the
ADP sensitivity at 0 °C, where the equilibration of the two pools is
slow. Fig. 8 presents results of
experiments in which the time course of dephosphorylation was monitored
following addition of 2.5 mM ADP together with 1 mM nonradioactive ATP to phosphoenzyme formed in the
presence of [-32P]ATP. The phosphorylation as well as
the dephosphorylation was carried out in the presence of 20 mM Na+ and absence of K+. Under
these conditions, E2P dephosphorylates only very
slowly, whereas E1P dephosphorylates rapidly
because of the reaction with ADP. Thus, the data points representing
the time course of dephosphorylation could be fitted by a biexponential
function where the extents of the rapid and slow decay components
reflect the initial amounts of the ADP-sensitive
E1P and ADP-insensitive
E2P, respectively. The dotted lines
in Fig. 8 show the extrapolation of the slow decay component back to
the ordinate intercept to indicate the amount of
E2P. For the wild type, the ordinate intercept
yielded about 80% E2P, indicating that the
steady-state distribution of the phosphoenzyme intermediates between
the E1P and E2P forms favored the ADP-insensitive E2P form. For
Thr214
Ala, the amount of E2P
was 60%. This is considerably more than the 25% previously observed
for Gly263
Ala (Table I). Hence, in contrast to
Gly263
Ala, the distribution of
E1P and E2P in
Thr214
Ala was only slightly more in favor of
E1P than in the wild type, in line with the
observation described above that the
E1-E2 conformational
equilibrium of the dephosphoenzyme was only slightly shifted in favor
of E1. From Fig. 8 it can, moreover, be seen that the rate constant corresponding to the slow phase, reflecting the
dephosphorylation of E2P was reduced in
Thr214
Ala relative to wild type. This reduction
amounted to about 2-fold relative to the wild-type enzyme (see legend
to Fig. 8).
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Dephosphorylation Kinetics at 25 °C of Phosphoenzyme Formed at
20 mM Na+--
The 2-fold reduction of the
E2P dephosphorylation rate seen in Fig. 8 raised
the question whether a slow dephosphorylation of
E2P is a general characteristic of
Thr214 Ala, observable also at a more physiological
temperature and in the presence of K+ to activate the
dephosphorylation. Consequently the E2P
dephosphorylation was studied at 25 instead of 0 °C, using the
quench-flow module as previously described (17, 33) and phosphorylation
conditions otherwise similar to the ADP sensitivity experiments to
promote the accumulation of E2P (presence of 20 mM NaCl with 130 mM choline chloride and
without K+, cf. Fig. 8). Fig.
9 shows that at 3 different sets of
dephosphorylation conditions: a Na+ concentration of 200 mM without K+ (Fig. 9A), a
nonsaturating K+ concentration of 1 mM (Fig.
9B), and a saturating K+ concentration of 20 mM (Fig. 9C), the dephosphorylation rate was
significantly reduced in the Thr214
Ala mutant relative
to wild type. The reduction amounted to 1.8- (Fig. 9A), 2.1- (Fig. 9B), and at least 4.8-fold (Fig. 9C). Because the dephosphorylation rate of the wild type at 20 mM K+ was too high to measure accurately (>300
s
1, Fig. 9C), the reduction seen for the
mutant in this condition may actually amount to more than 4.8-fold. It
is noteworthy that for all three conditions a monoexponential function
could be fitted satisfactorily to the data, indicating that at the
beginning of the dephosphorylation the major part of the phosphoenzyme
was present as E2P with very little admixture of
E1P. The reason that E2P
accumulated to such an extent is that at 25 °C in the presence of
only 20 mM Na+ without K+
(i.e. the conditions prevailing during formation of the
phosphoenzyme) the rate of E2P dephosphorylation
is more than 50-fold lower than the rates of phosphorylation and
E1P
E2P
interconversion, both for wild type and mutant (cf. Fig.
9A, where a rather low rate of E2P
dephosphorylation is seen even at 200 mM Na+
because of the absence of K+).
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Fig. 9 shows that a saturating K+ concentration of 20 mM enhanced the dephosphorylation rate 184- and 69-fold in
the wild type and the Thr214 Ala mutant, respectively,
relative to the rate in the mere presence of Na+. Hence,
the difference between the mutant and the wild type increased upon
binding of K+. For the Thr214
Ala mutant,
the dephosphorylation rate was more than half-maximal at 1 mM K+ (compare the rate constants of 38 and 69 s
1 obtained at 1 and 20 mM K+,
respectively), whereas for the wild type the dephosphorylation rate at
1 mM K+ was maximally 24% of that
corresponding to 20 mM K+, showing that the
reduced rate of E2P dephosphorylation in
Thr214
Ala is not caused by a decrease in the affinity
for K+.
Fig. 9C, furthermore, shows that the inhibition of the
dephosphorylation rate in Thr214 Ala could not be
overcome by increasing the Mg2+ concentration from 3 to 15 mM. For the wild type, the Mg2+ concentration
of 3 mM is saturating with respect to activation of
dephosphorylation. Likewise, the data obtained for the mutant at 15 mM Mg2+ were indistinguishable from those
obtained at 3 mM Mg2+, indicating that the
inhibition is not caused by a reduction of Mg2+ affinity.
Dephosphorylation Kinetics at 25 °C of Phosphoenzyme Formed at
600 mM NaCl--
Finally, the E1P
E2P interconversion was studied at 25 °C
(Fig. 10). To accumulate the
E1P form, phosphorylation was carried out in the
presence of a high NaCl concentration of 600 mM, which shifts the steady-state distribution of phosphoenzyme intermediates in
favor of E1P, as previously described (17, 33).
To observe the E1P
E2P conversion, dephosphorylation was initiated
by a downward jump in the Na+ concentration to 200 mM, and simultaneously 1 mM unlabeled ATP was
added together with 20 mM K+, followed by acid
quenching at various time intervals. The dephosphorylation involves the
steps E1P
E2P
E2. In the presence of a saturating K+ concentration of 20 mM,
E2P
E2 is much faster
than E1P
E2P in the
wild-type enzyme and does not contribute significantly to rate
limitation. Therefore, the wild type data could be satisfactorily fitted by a monoexponential decay function (rate constant 111 s
1, fit not shown). This was obviously not the case for
the mutant data (Fig. 10), the reason being that the
E1P
E2P transition in
the mutant is followed by a relatively slow E2P
E2 reaction. By fitting Equation 2 under
"Experimental Procedures," describing two consecutive first-order
reactions, with the rate constants for the dephosphorylation of
E2P set at 69 and 500 s
1 for
Thr214
Ala and the wild type, respectively, in
accordance with the data described above for Fig. 9C, the
respective rate constants corresponding to the
E1P
E2P transition
were found to be 57 and 139 s
1, indicating that the
E1P
E2P conversion
rate is reduced 2.4-fold in Thr214
Ala relative to wild
type.
|
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DISCUSSION |
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Thr214 Ala is the first
Na+,K+-ATPase mutant for which a change to the
intrinsic affinity of the E2 form for vanadate
and a, presumably related, inhibitory effect of the mutation on the
Vmax for dephosphorylation have been
demonstrated. These effects together with effects on
E1-E2 and
E1P-E2P conformational
equilibria all seem to contribute to the observed change in sensitivity
to vanadate inhibition of ATPase activity induced by the mutation.
Conformational Equilibria--
As summarized in Table I, titration
of the Na+, K+, and ATP dependence of ATPase
activity in Thr214 Ala showed changes relative to the
wild type compatible with a shift of the
E1-E2 conformational
equilibrium of the dephosphoenzyme in favor of
E1. This interpretation was further supported by
the K+-deocclusion analysis, demonstrating an increase of
the rate of the K+-deoccluding
E2(K2)
E1
step relative to wild type as well as a reduction of the level of the
E2(K2) intermediate in the absence of Na+ and ATP. Studies of the phosphoenzyme showed a
change in the steady-state distribution of ADP-sensitive
E1P and ADP-insensitive E2P in favor of E1P, and
the rate constant characterizing the E1P
E2P conversion was reduced in Thr214
Ala relative to wild type. Hence, it may be concluded that mutation
Thr214
Ala displaced the
E1-E2 and
E1P-E2P conformational
equilibria in parallel in favor of the E1 and
E1P forms, respectively.
Affinity for Vanadate--
A most remarkable feature displayed by
mutant Thr214 Ala was the conspicuous 150-fold
reduction of the apparent affinity for vanadate inhibition determined
in the ATPase assay (Fig. 3). The amount of enzyme-vanadate complex
formed depends on the concentration of the vanadate-reactive
E2 intermediate accumulated at steady state and
the intrinsic affinity of E2 for vanadate. The
displacement of the conformational equilibria in favor of the
E1 and E1P forms must,
therefore, have contributed to lower the apparent affinity for vanadate
through reduction of the steady-state concentration of
E2. It is noteworthy, however, that the
previously studied mutation Gly263
Ala in the loop
connecting domain A with membrane segment M3 (see Fig. 1)
exerted only a 7-fold reduction in the apparent vanadate affinity
relative to the wild type under the conditions prevailing during the
ATPase activity measurement (17), even though the displacement of the
conformational equilibria in favor of E1 and E1P was more pronounced than for
Thr214
Ala as judged on the basis of the apparent
affinities for Na+, K+, and ATP and the
analysis of K+ deocclusion and of the phosphoenzyme
intermediates (Table I). Consequently, the conspicuous reduction of
the apparent vanadate affinity observed for Thr214
Ala
cannot be attributed solely to the change of conformational equilibrium, and there must be additional contributing factors in this case.
Importantly, we were able to examine the intrinsic vanadate affinity of
the E2 form and the effect on the apparent
affinity for vanadate of displacement of the
E1-E2 equilibrium, using
for the first time a phosphorylation assay that determines the
vanadate-free enzyme fraction under equilibrium conditions
(i.e. in the absence of enzyme turnover producing
E2 from E2P) (Fig. 7).
For Gly263 Ala, the vanadate affinity determined by
this assay became wild type-like when the enzyme was forced into the
E2 form by the presence of the K+
congener Rb+, thus demonstrating that the intrinsic
vanadate affinity of E2 is normal in this
mutant. By contrast, Thr214
Ala retained a reduced
affinity for vanadate in the E2 form accumulated
in the presence of Rb+, indicating that Thr214
Ala manifests true low affinity for vanadate.
Defective Catalysis of E2P Dephosphorylation--
In
dephosphorylation experiments with phosphoenzyme accumulated under
conditions favoring the E2P form,
Thr214 Ala showed a reduced dephosphorylation rate
relative to wild type. This was seen independently of whether
dephosphorylation was activated by Na+ or by nonsaturating
or saturating K+ concentrations (Fig. 9). Normally,
K+ acting at extracellularly facing sites far away from the
catalytic site activates the dephosphorylation, and the reduced
dephosphorylation rate in Thr214
Ala could possibly
involve defective K+ binding. However, the difference
between Thr214
Ala and the wild type with respect to
the dephosphorylation rate increased when the K+
concentration was increased from 1 to 20 mM, and the
dephosphorylation rate of the mutant was more than half-maximal at 1 mM K+, whereas the dephosphorylation rate
displayed by the wild type at this K+ concentration was
maximally 24% of the rate at 20 mM K+.
Therefore, the inhibition of E2P
dephosphorylation in Thr214
Ala must be brought about
by a reduction in Vmax for dephosphorylation rather than by a decrease in K+ affinity at the activating
E2P sites. Such an inhibitory effect on the
Vmax for dephosphorylation has not previously
been reported for any Na+,K+-ATPase mutant.
Together with the 2.4-fold reduction of the E1P
E2P conversion rate, the reduction of the
E2P dephosphorylation rate, corresponding to at
least 4.8-fold at 20 mM K+, can account for the
low turnover rate displayed by the mutant (38% of wild type). It is,
furthermore, likely that the reduced rate of production of
E2 caused by the inhibition of dephosphorylation contributes to the striking reduction of the apparent vanadate affinity
observed in the ATPase assay, through reduction of the steady-state
concentration of E2.
Our observations can be explained according to the classic concept that
the catalytic rate (in this case the rate of E2P
dephosphorylation) depends on the ability of the enzyme to bind the
activated complex (transition state) tightly, thereby lowering the
energy barrier that has to be traversed during the reaction (35, 36).
Hence, the catalytic rate increases with an increase of the affinity of
the enzyme for the transition state complex, and decreases in cases
where the transition state complex is bound less tightly. Because
vanadate is considered an analog of the pentacoordinated transition
state of the phosphoryl group (34), it is likely that the reduced
intrinsic affinity of the E2 form for vanadate in Thr214 Ala is directly related to the inhibition of
E2P dephosphorylation, implying that defective
binding of the phosphoryl transition state complex (transition state
destabilization) causes the inability to catalyze
E2P dephosphorylation properly.
The catalytic site performs very different functions in
E2/E2P as compared with
E1/E1P ("phosphatase"
and "kinase" functions, respectively). In
E2P, a nucleophilic attack by water on the
aspartyl phosphate bond is facilitated. It is notable that in
Thr214 Ala the catalytic mechanism is defective only in
E2/E2P, the phosphorylation of E1 from ATP being wild
type-like (Fig. 5). This supports a mechanism where the conserved
214TGES of domain A is isolated in the
E1 form, but makes contact with the
phosphorylation site and becomes catalytically important in the
E2/E2P conformations
(cf. Fig. 1). It is interesting to note the analogy to the
non-ATPase members of the family of aspartyl-phosphate-utilizing phosphohydrolases/phosphotransferases such as phosphoserine
phosphatase, which recently was studied by x-ray crystallography in
several intermediates (37). These enzymes have in common with the
P-type ATPases several highly conserved residues of the Rossmann fold corresponding to domain P, but lack domain A with
the TGES sequence present in P-type ATPases. Nevertheless, the
catalytic function of phosphoserine phosphatase requires participation
of residues such as Glu20 outside the Rossmann fold, to
assist the nucleophilic attack and stabilize the transition state (37).
In the P-type ATPases, the corresponding requirement for domain
A residues for proper catalysis of
E2P hydrolysis, and the ability of domain
A to move as shown in Fig. 1, thereby initiating
conformational rearrangements in the membrane, may constitute an
important element in the coupling of substrate utilization with cation transport.
The defective catalysis of E2P dephosphorylation
in Thr214 Ala would be in accordance with a role of the
side chain hydroxyl of Thr214 in the positioning of the
attacking water molecule or in direct interaction with the phosphate in
the transition state, as well as a more indirect role in optimization
of the structure of the catalytic site in the
E2P form through hydrogen bonding to other residues. Hydrogen bond formation with residues in domain P
could also explain the importance of Thr214 in the
E1-E2 and
E1P-E2P conformational
changes, where domain A is thought to dock into domain
P (cf. Fig. 1). The side chain interactions
could, furthermore, affect the orientation of the main chain carbonyl
group corresponding to Thr214, which in the crystal
structure of the Ca2+-ATPase in the
E2 form is rather close to other catalytically important residues such as Asp703 (Asp712 in
Na+,K+-ATPase2).
Mg2+ or another divalent cation such as Fe2+ is
required as cofactor for the phosphorylation of
E1 by ATP and for the hydrolysis of
E2P (1). On the basis of detection of
Fe2+-catalyzed oxidative cleavage near the TGES sequence in
E2 and E2P, but not in
the E1 form, Karlish and co-workers (38) have proposed that the glutamate, Glu216, of TGES contributes to
ligation of Mg2+ in E2 and
E2P, whereas in E1 and
E1P the coordination has changed so that all
Mg2+ coordinating groups now come from the P and
N domains. In analogy with phosphoserine phosphatase (37),
Mg2+ ligands might be contributed by P-domain
residues Asp371, Thr373, and Asp712
(39) (numbering corresponding to
Na+,K+-ATPase2). In phosphoserine
phosphatase, the remaining three Mg2+ ligands are a
phosphate oxygen and two water molecules (37). It is possible that in
P-type ATPases one or both water molecules are substituted by protein
groups of domain A in E2 and
E2P, but not in E1 and
E1P, or that protein groups of domain
A serve to position the Mg2+ liganding water
molecules in E2 and E2P.
In accordance with a crucial function of the glutamate of TGES, we
found that the Na+,K+-ATPase mutant
Glu216 Ala is unable to maintain cell viability. This
indicates that the transport rate or the expression level is very low
in Glu216
Ala. The advantage with the
Thr214
Ala mutant was that although the function of
this mutant clearly was defective, the activity and expression levels
were sufficiently high to allow the detailed investigation of both the
overall and the partial reactions. It is presently not known whether
substitution of Thr214 with residues other than alanine
would lead to similar or quite different functional consequences as
observed for Thr214
Ala. In principle, the mechanism
underlying the inhibition of E2P
dephosphorylation in Thr214
Ala could involve a defect
in the ligation of the catalytic Mg2+ ion. The catalytic
ability of mutant Thr214
Ala was, however, not improved
by a 5-fold increase of the Mg2+ concentration (Fig.
9C). Thus, if Mg2+ binding is defective in the
mutant, it must be the positioning of the bound Mg2+ ion
and not simply the affinity, which is changed.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Jens Peter Andersen for discussion and many helpful suggestions, Jytte Jørgensen, Janne Petersen, and Kirsten Lykke Pedersen for expert technical assistance, and Dr. R. J. Kaufman, Genetics Institute, Boston, MA, for the expression vector pMT2.
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FOOTNOTES |
---|
* This work was supported by grants from the Danish Medical Research Council, the Research Foundation of Aarhus University, the Novo Nordisk Foundation, Denmark, and the Lundbeck Foundation, Denmark.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 Physiology,
University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C,
Denmark. Fax: 45-86-12-90-65; E-mail: bv@fi.au.dk.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212136200
2
All numbering of
Na+,K+-ATPase residues in this article refers
to the sequence of the rat 1 isoform.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Na+, K+-ATPase, the Na+- and K+-transporting adenosine triphosphatase (EC 3.6.1.37); E1 and E2, conformational states of the Na+,K+-ATPase; E1P and E2P, phosphorylated conformational states; K0.5, ligand concentration giving half-maximum activation or inhibition; M1-M10, putative transmembrane segments numbered from the NH2-terminal end of the peptide chain.
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