Importance of Conserved Thr214 in Domain A of the Na+,K+-ATPase for Stabilization of the Phosphoryl Transition State Complex in E2P Dephosphorylation*

Mads Toustrup-Jensen and Bente VilsenDagger

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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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 right-arrow Ala. Thr214 right-arrow 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 right-arrow Ala retained a reduced vanadate affinity relative to wild type, whereas the affinity of Gly263 right-arrow Ala became wild type-like. Thus, mutation of Thr214 affected the intrinsic affinity of E2 for vanadate. Furthermore, Thr214 right-arrow 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 right-arrow 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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) right-arrow E2P transition. The countertransport of K+ is associated with the dephosphorylation of E2P and another conformational change, E2(K2) right-arrow 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 alpha -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 right-arrow E2(K2) and E1P(Na3) right-arrow E2P transitions, thereby displacing domain N from domain P (14). It was hypothesized that the E1P(Na3) right-arrow 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) right-arrow E2P transition (15). Other residues in domain A have also been shown by mutagenesis to be involved in the E2(K2) right-arrow E1 and E1P(Na3) right-arrow E2P transitions of the Na+,K+-ATPase. Hence, mutation of Glu233 in the Na+,K+-ATPase accelerated the E2(K2) right-arrow 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) right-arrow E1 and E1P(Na3) right-arrow 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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Fig. 1.   Structural model based on the Ca2+-ATPase crystal structures, showing movement of domains A and P in connection with the E1-E2 transition. In the E2 form, Thr214 in domain A approaches the phosphorylated aspartate, Asp371, in domain P. The left and right panels show the E1Ca2 state and the Ca2+-free E2 state of the Ca2+-ATPase, respectively (Protein Data Bank accession codes 1EUL and 1IWO). The indicated numbering of residues corresponds to the Na+,K+-ATPase rat alpha 1 isoform. The residues Thr214 (mutated in the present work), Asp371 (the phosphorylated aspartate), and Gly263 (studied previously (17)) are highlighted in yellow, red, and white, respectively. Domains A, P, and N are indicated and shown in different colors. Prepared by use of the WebLab ViewerPro program (Molecular Simulations Ltd., Cambridge, United Kingdom).

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 right-arrow Ala mutant. The effects of the Thr214 right-arrow 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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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Mutagenesis, Expression, and Basic Functional Characterization-- Oligonucleotide-directed mutagenesis (25) of the cDNA encoding the ouabain-insensitive rat alpha 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 [gamma -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 [gamma -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 [gamma -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 [gamma -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,


E<UP>P=</UP>(E<UP>P<SUB>max</SUB>−</UP>E<UP>P<SUB>∞</SUB></UP>)<UP>·</UP>(<UP>1−</UP>[<UP>vanadate</UP>]<UP>/</UP>(K<SUB><UP>0.5</UP></SUB><UP>+</UP>[<UP>vanadate</UP>]))<UP>+</UP>E<UP>P<SUB>∞</SUB></UP> (Eq. 1)
EPmax is the phosphorylation level obtained in the absence of vanadate, and EPinfinity corresponds to infinite vanadate concentration.

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 right-arrow 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 right-arrow E2P right-arrow E2 with rate constants k1 and k2, respectively,
E<UP>P</UP><SUB>t</SUB>=[E<SUB><UP>1</UP></SUB><UP>P</UP>]<UP> + </UP>[E<SUB><UP>2</UP></SUB>P]=E<UP>P<SUB>0</SUB> · </UP><FENCE><FR><NU>k<SUB><UP>2</UP></SUB></NU><DE>k<SUB><UP>2</UP></SUB>−k<SUB><UP>1</UP></SUB></DE></FR><UP>e</UP><SUP><UP>−</UP>k<SUB><UP>1</UP></SUB>t</SUP>−<FR><NU>k<SUB><UP>1</UP></SUB></NU><DE>k<SUB><UP>2</UP></SUB><UP>−</UP>k<SUB><UP>1</UP></SUB></DE></FR><UP>e</UP><SUP><UP>−</UP>k<SUB><UP>2</UP></SUB>t</SUP></FENCE> (Eq. 2)
where EPt is the phosphorylation level at time t and EP0 is the initial phosphorylation level.

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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INTRODUCTION
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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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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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Fig. 2.   Na+, K+, and ATP dependence of ATPase activity. The rate of ATP hydrolysis was determined for the expressed wild-type Na+,K+-ATPase (filled circles) and mutant Thr214 right-arrow Ala (open triangles) at 37 °C in the presence of 30 mM histidine buffer (pH 7.4), 1 mM EGTA, 10 µM ouabain, 3 mM MgCl2, and (A) 20 mM KCl, 3 mM ATP, and the indicated concentrations of NaCl, (B) 40 mM NaCl, 3 mM ATP, and the indicated concentrations of KCl, or (C) 130 mM NaCl, 20 mM KCl, and the indicated concentrations of ATP. The data points are average values corresponding to three independent titrations. Error bars can be seen when larger than the size of the symbol. Each line shows the best fit of the Hill equation, resulting in the K0.5 values listed in Table I.


                              
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Table I
Comparison of conformational equilibria of wild type, Thr214 right-arrow Ala, and Gly263 right-arrow Ala

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 min-1 was found for Thr214 right-arrow 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 right-arrow 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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Fig. 3.   Vanadate dependence of Na+,K+-ATPase activity. The rate of ATP hydrolysis was determined for the expressed wild-type Na+,K+-ATPase (filled circles) and mutant Thr214 right-arrow Ala (open triangles) at 37 °C in the presence of 130 mM NaCl, 20 mM KCl, 3 mM ATP, 3 mM MgCl2, 30 mM histidine buffer (pH 7.4), 1 mM EGTA, 10 µM ouabain, and the indicated concentrations of vanadate. Average values corresponding to four to nine independent titrations are shown as percentage of the Na+,K+-ATPase activity measured in the absence of vanadate. Error bars can be seen when larger than the size of the symbol. Each line shows the best fit of the Hill equation. The corresponding K0.5 values are 2.5 and 374 µM for the wild type and the Thr214 right-arrow Ala mutant, respectively.

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 right-arrow 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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Fig. 4.   Na+ dependence of phosphorylation from ATP. Phosphorylation was carried out with the wild-type Na+,K+-ATPase (filled circles) and mutant Thr214 right-arrow Ala (open triangles) for 15 s at 0 °C in the presence of 20 mM Tris (pH 7.5), 3 mM MgCl2, 1 mM EGTA, 2 µM [gamma -32P]ATP, 10 µM ouabain, oligomycin (20 µg/ml), and various concentrations of NaCl as indicated on the abscissa, with N-methyl-D-glucamine to keep the ionic strength constant at 150 mM. The data points are average values corresponding to four to six independent titrations. Error bars can be seen when larger than the size of the symbol. Each line shows the best fit of the Hill equation. The corresponding K0.5 values are 0.55 and 0.34 mM for the wild type and Thr214 right-arrow Ala mutant, respectively.

Time Course of Phosphorylation with [gamma -32P]ATP-- To test the phosphorylation reaction E1Na3 right-arrow E1P(Na3) and the ATP affinity of the E1Na3 form, the time course of phosphorylation with 2 µM [gamma -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 right-arrow 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 right-arrow Ala the phosphorylation reaction as well as the binding of ATP to E1Na3 is wild type-like.


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Fig. 5.   Time course of phosphorylation by [gamma -32P]ATP at 25 °C. Rapid kinetic measurements at 25 °C of the time course of phosphorylation were performed with the wild-type Na+,K+-ATPase (filled circles) and the mutant Thr214 right-arrow Ala (open triangles) in the presence of 100 mM NaCl, 40 mM Tris (pH 7.5), 3 mM MgCl2, 1 mM EGTA, 10 µM ouabain, oligomycin (20 µg/ml), and 2 µM [gamma -32P]ATP, using the QFM-5 module according to Protocol 1 (17). The data points are average values of three experiments. Error bars can be seen when larger than the size of the symbol. Each line shows the best fit of a monoexponential time function. The rate constants are 29 and 32 s-1, and the maximum level of phosphorylation reached, indicated as 100%, constituted 96, and 94% of the active site concentration, for the wild type and the Thr214 right-arrow Ala mutant, respectively.

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) right-arrow 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 [gamma -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) right-arrow E1 right-arrow E1Na3 right-arrow 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 right-arrow 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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Fig. 6.   Time course of K+ deocclusion. Measurements were performed with the wild-type Na+,K+-ATPase (filled circles) and the mutant Thr214 right-arrow Ala (open triangles). Enzyme treated with 20 µM ouabain, 3 mM MgCl2, and 20 mM Tris buffer (pH 7.5) was equilibrated for 1 h at room temperature with 8 mM K+. Oligomycin (150 µg/ml) was then added, and the solution was cooled to 10 °C and diluted 10-fold by addition of a phosphorylation solution of the same temperature, producing final concentrations of 1 µM [gamma -32P]ATP, 100 mM NaCl, 1 mM MgCl2, 1 mM EGTA, and 20 mM Tris (pH 7.5). The phosphorylation was monitored by acid quenching at various time intervals. For determination of the 100% value representing the fully deoccluded enzyme, the 1-h incubation was carried out in the presence of 50 mM Na+ and absence of K+. Average values corresponding to two to seven independent experiments were analyzed by fitting a biphasic time function as previously described (17, 31). The slow component is indicated by the line, and the extracted rate constant, reflecting K+ deocclusion, and its amplitude, corresponding to the relative amount of enzyme initially present as E2(K2), are listed in Table I. Error bars can be seen when larger than the size of the symbol.

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 [gamma -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 [gamma -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. 7.   Vanadate binding at equilibrium in the absence and presence of Rb+. Wild-type Na+,K+-ATPase (filled circles), Thr214 right-arrow Ala (open triangles), or Gly263 right-arrow Ala (open squares) was equilibrated at 20 °C for 30 min in the presence of the indicated concentrations of orthovanadate in the absence (A) or presence (B, main part) of 8 mM RbCl. Following cooling of the samples to 0 °C the enzyme fraction with no vanadate bound was determined by its ability to form a phosphoenzyme upon the addition of [gamma -32P]ATP as described under "Experimental Procedures." The data points are average values corresponding to four to eight independent experiments, calculated following normalization to the 100% value obtained in the absence of vanadate. The lines show the best fits of Equation 1 (see "Experimental Procedures"), resulting in the K0.5 values listed in Table II. The inset shows deocclusion experiments (performed according to the principles described for Fig. 6) in the absence of vanadate, but under conditions otherwise similar to those prevailing in the vanadate binding assay, to determine the fraction of the enzyme present as E2(Rb2) (100% minus the ordinate intercept) and demonstrate the fraction of the vanadate-free enzyme that is phosphorylated after 30 s (i.e. the 100% value in the main part). The enzyme fraction present as E2(Rb2) was 100, 100, and 89%, for wild type, Thr214 right-arrow Ala, and Gly263 right-arrow Ala, respectively. The data points in the inset are average values corresponding to three to four independent experiments. Error bars can be seen when larger than the size of the symbol.


                              
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Table II
Equilibrium binding affinities for vanadate determined in phosphorylation experiments

Fig. 7A shows results obtained following equilibration with vanadate in the absence of any of the transported cations. Under these conditions, Thr214 right-arrow 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 right-arrow Ala (17) that exhibited more pronounced changes than Thr214 right-arrow 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 right-arrow Ala as compared with Thr214 right-arrow Ala, the apparent affinity for vanadate observed in the absence of the transported cations (Fig. 7A) was also reduced more in Gly263 right-arrow Ala (22-fold reduction relative to wild type) than in Thr214 right-arrow Ala.

An important question was, however, whether the relatively small change to the E1-E2 conformational equilibrium of Thr214 right-arrow 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 right-arrow 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 right-arrow 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 [gamma -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 right-arrow Ala, the amount of E2P was 60%. This is considerably more than the 25% previously observed for Gly263 right-arrow Ala (Table I). Hence, in contrast to Gly263 right-arrow Ala, the distribution of E1P and E2P in Thr214 right-arrow 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 right-arrow 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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Fig. 8.   Time course of ADP-dependent dephosphorylation at 0 °C. The wild-type Na+,K+-ATPase (filled circles) and mutant Thr214 right-arrow Ala (open triangles) were phosphorylated for 10 s at 0 °C in the presence of 2 µM [gamma -32P]ATP, 20 mM NaCl, 130 mM choline chloride, 20 mM Tris (pH 7.5), 3 mM MgCl2, 1 mM EGTA, and 10 µM ouabain. Dephosphorylation was monitored by addition of a chase solution producing a final concentration of 1 mM unlabeled ATP and 2.5 mM ADP, followed by acid quenching at the indicated time intervals. The data points are average values corresponding to five to eight independent experiments, calculated following normalization to the 100% value obtained by quenching after 10 s of phosphorylation without dephosphorylation. Error bars can be seen when larger than the size of the symbol. The lines show the best fit of a biexponential time function. Note the logarithmic ordinate scale. The dotted lines show the extrapolation of the slow decay component corresponding to E2P back to the ordinate intercept to show its initial value. The extents of the rapid and slow decay components corresponding to the initial values of E1P and E2P, respectively, are given in Table I. The rate constant corresponding to the slow decay component (E2P hydrolysis) is 0.034 s-1 for wild type, and 0.018 s-1 for Thr214 right-arrow Ala.

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 right-arrow 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 right-arrow 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 right-arrow 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.   Dephosphorylation of phosphoenzyme formed at 20 mM NaCl. Rapid kinetic measurements at 25 °C of dephosphorylation of phosphoenzyme formed in the presence of 20 mM NaCl, 2 µM [gamma -32P]ATP, 130 mM choline chloride, 20 mM Tris (pH 7.5), 3 mM MgCl2, 1 mM EGTA, and 10 µM ouabain were carried out with the wild-type Na+,K+-ATPase (filled circles) and Thr214 right-arrow Ala (open triangles) using the QFM-5 module according to Protocol 2b as described previously (33). Dephosphorylation was monitored by addition of a chase solution producing final concentrations of 1 mM unlabeled ATP and (A) 200 mM NaCl, 65 mM choline chloride, and 3 mM MgCl2, (B) 1 mM KCl, 20 mM NaCl, 130 mM choline chloride, and 3 mM MgCl2, and (C) 20 mM KCl, 20 mM NaCl, 130 mM choline chloride, and 3 mM MgCl2 (open triangles pointing upwards) or 15 mM MgCl2 (open triangles pointing downwards), followed by acid quenching at the indicated time intervals. The data points are average values corresponding to two to three independent experiments, calculated following normalization to the 100% value obtained by quenching after 5 s of phosphorylation without dephosphorylation. Error bars can be seen when larger than the size of the symbol. Each line shows the best fit of a monoexponential time function giving the following rate constants: (A) wild type, 1.8 s-1; Thr214 right-arrow Ala, 1.0 s-1; (B) wild type, 80 s-1; Thr214 right-arrow Ala, 38 s-1 ; (C) wild type, 331 s-1; Thr214 right-arrow Ala, 69 s-1 (open triangles pointing upwards) or 71 s-1 (open triangles pointing downwards).

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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow E2P right-arrow E2. In the presence of a saturating K+ concentration of 20 mM, E2P right-arrow E2 is much faster than E1P right-arrow 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 right-arrow E2P transition in the mutant is followed by a relatively slow E2P right-arrow 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 right-arrow Ala and the wild type, respectively, in accordance with the data described above for Fig. 9C, the respective rate constants corresponding to the E1P right-arrow E2P transition were found to be 57 and 139 s-1, indicating that the E1P right-arrow E2P conversion rate is reduced 2.4-fold in Thr214 right-arrow Ala relative to wild type.


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Fig. 10.   Dephosphorylation of phosphoenzyme formed at 600 mM NaCl. Rapid kinetic measurements at 25 °C of dephosphorylation of phosphoenzyme formed in the presence of 600 mM NaCl, 2 µM [gamma -32P]ATP, 20 mM Tris (pH 7.5), 3 mM MgCl2, 1 mM EGTA, and 10 µM ouabain were carried out with the wild-type Na+,K+-ATPase (filled circles) and Thr214 right-arrow Ala (open triangles) using the QFM-5 module according to Protocol 2a as described previously (33). Dephosphorylation was monitored by addition of a chase solution producing final concentrations of 200 mM NaCl, 1 mM unlabeled ATP, and 20 mM KCl, followed by acid quenching at the indicated time intervals. The data points are average values corresponding to three independent experiments, calculated following normalization to the 100% value obtained by quenching after 5 s of phosphorylation without dephosphorylation. Error bars can be seen when larger than the size of the symbol. Each line shows the best fit of Equation 2 (see "Experimental Procedures") with the rate constant corresponding to E2P dephosphorylation set at 500 and 69 s-1 for wild type and Thr214 right-arrow Ala, respectively. The extracted rate constants corresponding to the E1P right-arrow E2P conversion are 139 and 57 s-1 for wild type and Thr214 right-arrow Ala, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thr214 right-arrow 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 right-arrow 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) right-arrow 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 right-arrow E2P conversion was reduced in Thr214 right-arrow Ala relative to wild type. Hence, it may be concluded that mutation Thr214 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow Ala retained a reduced affinity for vanadate in the E2 form accumulated in the presence of Rb+, indicating that Thr214 right-arrow 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 right-arrow 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 right-arrow Ala could possibly involve defective K+ binding. However, the difference between Thr214 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow 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 right-arrow Ala is unable to maintain cell viability. This indicates that the transport rate or the expression level is very low in Glu216 right-arrow Ala. The advantage with the Thr214 right-arrow 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 right-arrow Ala. In principle, the mechanism underlying the inhibition of E2P dephosphorylation in Thr214 right-arrow Ala could involve a defect in the ligation of the catalytic Mg2+ ion. The catalytic ability of mutant Thr214 right-arrow 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.

    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.

    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.

Dagger 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 alpha 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Glynn, I. M. (1985) in The Enzymes of Biological Membranes (Martonosi, A. N., ed), Vol. 3 , pp. 35-114, Plenum Publishing Corp., New York
2. Albers, R. W. (1967) Annu. Rev. Biochem. 36, 727-756
3. Post, R. L., Kume, S., Tobin, T., Orcutt, B., and Sen, A. K. (1969) J. Gen. Physiol. 54, 306s-326s
4. Post, R. L., Hegyvary, C., and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540[Abstract/Free Full Text]
5. Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655[CrossRef][Medline] [Order article via Infotrieve]
6. Sweadner, K. J., and Donnet, C. (2001) Biochem. J. 356, 685-704[CrossRef][Medline] [Order article via Infotrieve]
7. Hebert, H., Purhonen, P., Vorum, H., Thomsen, K., and Maunsbach, A. B. (2001) J. Mol. Biol. 314, 479-494[CrossRef][Medline] [Order article via Infotrieve]
8. Lingrel, J. B., and Kuntzweiler, T. (1994) J. Biol. Chem. 269, 19659-19662[Free Full Text]
9. MacLennan, D. H., Rice, W. J., and Green, N. M. (1997) J. Biol. Chem. 272, 28815-28818[Free Full Text]
10. Vilsen, B., Ramlov, D., and Andersen, J. P. (1997) Ann. N. Y. Acad. Sci. 834, 297-309[Medline] [Order article via Infotrieve]
11. Jorgensen, P. L., and Pedersen, P. A. (2001) Biochim. Biophys. Acta 1505, 57-74[Medline] [Order article via Infotrieve]
12. Møller, J. V., Juul, B., and le Maire, M. (1996) Biochim. Biophys. Acta 1286, 1-51[Medline] [Order article via Infotrieve]
13. Axelsen, K. B., and Palmgren, M. G. (1998) J. Mol. Evol. 46, 84-101[Medline] [Order article via Infotrieve]
14. Goldshleger, R., Patchornik, G., Shimon, M. B., Tal, D. M., Post, R. L., and Karlish, S. J. D. (2001) J. Bioenerg. Biomembr. 33, 387-399[CrossRef][Medline] [Order article via Infotrieve]
15. Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 14088-14092[Abstract/Free Full Text]
16. Boxenbaum, N., Daly, S. E., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998) J. Biol. Chem. 273, 23086-23092[Abstract/Free Full Text]
17. Toustrup-Jensen, M., Hauge, M., and Vilsen, B. (2001) Biochemistry 40, 5521-5532[CrossRef][Medline] [Order article via Infotrieve]
18. Andersen, J. P., Vilsen, B., Leberer, E., and MacLennen, D. H. (1989) J. Biol. Chem. 264, 21018-21023[Abstract/Free Full Text]
19. Sørensen, T. L., Dupont, Y., Vilsen, B., and Andersen, J. P. (2000) J. Biol. Chem. 275, 5400-5408[Abstract/Free Full Text]
20. Danko, S., Yamasaki, K., Daiho, T., Suzuki, H., and Toyoshima, C. (2001) FEBS Lett. 505, 129-135[CrossRef][Medline] [Order article via Infotrieve]
21. Møller, J. V., Lenoir, G., Marchand, C., Montigny, C., Le Maire, M., Toyoshima, C., Juul, B. S., and Champeil, P. (2002) J. Biol. Chem. 277, 38647-38659[Abstract/Free Full Text]
22. Jørgensen, P. L., and Andersen, J. P. (1988) J. Membr. Biol. 103, 95-120[Medline] [Order article via Infotrieve]
23. Andersen, J. P., Vilsen, B., Collins, J. H., and Jørgensen, P. L. (1986) J. Membr. Biol. 93, 85-92[Medline] [Order article via Infotrieve]
24. Toyoshima, C., and Nomura, H. (2002) Nature 418, 605-611[CrossRef][Medline] [Order article via Infotrieve]
25. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 85, 3314-3318
26. Vilsen, B. (1992) FEBS Lett. 314, 301-307[CrossRef][Medline] [Order article via Infotrieve]
27. Vilsen, B. (1993) Biochemistry 32, 13340-13349[Medline] [Order article via Infotrieve]
28. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
29. Vilsen, B. (1999) Biochemistry 38, 11389-11400[CrossRef][Medline] [Order article via Infotrieve]
30. Vilsen, B. (1997) Biochemistry 36, 13312-13324[CrossRef][Medline] [Order article via Infotrieve]
31. Vilsen, B., and Andersen, J. P. (1998) Biochemistry 37, 10961-10971[CrossRef][Medline] [Order article via Infotrieve]
32. Smith, R. L., Zinn, K., and Cantley, L. C. (1980) J. Biol. Chem. 255, 9852-9859[Abstract/Free Full Text]
33. Toustrup-Jensen, M., and Vilsen, B. (2002) J. Biol. Chem. 277, 38607-38617[Abstract/Free Full Text]
34. Cantley, L. C., Jr., Cantley, L. G., and Josephson, L. (1978) J. Biol. Chem. 253, 7361-7368[Medline] [Order article via Infotrieve]
35. Pauling, L. (1946) Chem. Eng. News. 24, 1375-1377
36. Fersht, A. (1985) Enzyme Structure and Mechanism , 2nd Ed. , pp. 1-475, W. H. Freeman and Company, New York
37. Wang, W., Cho, H. S., Kim, R., Jancarik, J., Yokota, H., Nguyen, H. H., Grigoriev, I. V., Wemmer, D. E., and Kim, S.-H. (2002) J. Mol. Biol. 319, 421-431[CrossRef][Medline] [Order article via Infotrieve]
38. Patchornik, G., Goldshleger, R., and Karlish, S. J. D. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11954-11959[Abstract/Free Full Text]
39. Clausen, J. D., McIntosh, D. B., Woolley, D. G., and Andersen, J. P. (2001) J. Biol. Chem. 276, 35741-35750[Abstract/Free Full Text]


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