Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

Abstract

The segment708TGDGVNDSPALKK720 in the α-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg2+ or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr708 → Ser mutation and alterations of Asp714 abolish all catalytic reactions. In mutations of Asp710 and Asn713, ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp710contributes to coordination of Mg2+ during transfer of γ-phosphate to Asp369 in the high energy Mg·E1P[3Na] intermediate and that Asn713 is involved in these processes. In contrast, Asp710 and Asp713 do not contribute to Mg2+ binding in the E2P·ouabain complex. Transition to E2P thus involves a shift of Mg2+ coordination away from Asp710 and Asn713, and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp369. The Asp710 → Ala mutation blocks interaction with vanadate, whereas Asn713 → Ala interferes with phosphorylation from Pi of the E2·ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp710 → Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl+ 2- to 3-fold, suggesting a role in transmission of K+ stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain. The segment708TGDGVNDSPALKK720 in the α-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg2+ or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr708 → Ser mutation and alterations of Asp714 abolish all catalytic reactions. In mutations of Asp710 and Asn713, ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp710contributes to coordination of Mg2+ during transfer of γ-phosphate to Asp369 in the high energy Mg·E1P[3Na] intermediate and that Asn713 is involved in these processes. In contrast, Asp710 and Asp713 do not contribute to Mg2+ binding in the E2P·ouabain complex. Transition to E2P thus involves a shift of Mg2+ coordination away from Asp710 and Asn713, and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp369. The Asp710 → Ala mutation blocks interaction with vanadate, whereas Asn713 → Ala interferes with phosphorylation from Pi of the E2·ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp710 → Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl+ 2- to 3-fold, suggesting a role in transmission of K+ stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain. 5′-(p-fluorosulfonyl)benzoyladenosine loop between transmembrane segments 2 and 3 in α-subunit of Na,K-ATPase transmembrane segment 5 of 10 segments in α-subunit of Na,K-ATPase 4-morpholinepropanesulfonic acid wild type In resting mammals, Na,K-ATPase transforms 20–30% of the current ATP production to active Na,K transport in kidney, the central nervous system, and other cells of the body in which Na,K gradients are required for maintaining membrane potential, cell volume, and secondary active transport processes of other solutes. In the renal Na,K-pump, α1-subunits (1016 residues) and β1-subunits (302 residues) exist in a stoichiometric ratio of one (1Jorgensen P.L. De Pont J.J.H.H.M. Molecular Aspects of Transport Proteins. Elsevier, New York1992: 1-26Google Scholar). Long range structural E1 ↔ E2 transitions in the protein mediate energy conversion between the ATP binding and phosphorylation domain in the large cytoplasmic loop and cation sites in membrane-spanning segments of the α-subunit (2Jorgensen P.L. Andersen J.P. J. Membr. Biol. 1988; 103: 95-120Crossref PubMed Scopus (269) Google Scholar, 3Vilsen B. Andersen J.P. Petersen J. Jorgensen P.L. J. Biol. Chem. 1987; 262: 10511-10517Abstract Full Text PDF PubMed Google Scholar, 4Lingrel J.B Kuntzweiler T.A. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar) (Fig.1). Among P-type E1 → E2 ATPase pumps of widely different cation specificities, the sequence following the transiently phosphorylated residue Asp369 (369DKTGTLT) and the709GDGNDPL sequence with Asp710, Asn713, and Asp714 are highly conserved during evolution (5Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (735) Google Scholar). In agreement with this extensive homology, the recent appearance of the high resolution structure of Ca-ATPase (6Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar) shows that the TGDGVND segment is important for transfer of energy to the cation sites. It is located in the P domain with connections to M4 and M5, and the Asp710 side chain is the neighbor to the transiently phosphorylated residue Asp369 in a negatively charged surface of the domain. However, the role of the TGDGVND segment for binding of ATP or Mg2+ and its participation in the conformational transitions remain uncertain. The aim of the present work has been to establish structure-function relationships for the708TGDGVNDSPALKK720 segment, in particular concerning its role in coordination of Mg2+ or in binding of free ATP.Figure 6Effect of mutations of Asp710 or Asn713 on Mg2+ dependence of [3H]ouabain binding in phosphate or vanadate medium.Aliquots of about 200 μg of SDS-treated membranes were incubated for 60 min at 37 °C with 0.001–3 mm MgCl2, 1 mm EDTA-Tris, 3 mm Tris-phosphate, or 1 mm Tris-vanadate, 10 mm MOPS-Tris, pH 7.2, and [3H]ouabain (Amersham Pharmacia Biotech, specific activity 36 Ci/mmol) and cold ouabain to final concentrations of 10 nm for wild type (●), 10 or 40 nm for D713A (♦), and 90 nm for D710A (▴). Separation of free and bound [3H]ouabain was done as described under “Experimental Procedures.” Free Mg2+ concentrations were calculated for the Mg2+-EDTA buffer system using the Bad Chelator program (23Marks P.W. Maxfield F.R. Anal. Biochem. 1991; 193: 61-71Crossref PubMed Scopus (92) Google Scholar). For each Mg2+ concentration, ouabain binding was estimated in percentage of the maximum binding capacity that was determined in parallel for each SDS-treated preparation. The lines were fitted and K0.5(Mg2+) values were estimated using the binding a in which a is the maximum ouabain binding is and is the are in of mutations of Asp710 or Asn713 for Tl+ and K+ of ATP binding is expressed as the ratio of Tl+ of to [3H]ouabain binding capacity in each are values of two were as described P.L. 1998; PubMed Scopus Google Scholar). The lines were fitted using the binding ratio a a is the maximum Tl+ binding is and is the The values were for wild type and for a and 11 for D710A a and for a and and for a and 2 For K+ of ATP membranes were incubated with nm in the presence of mm was to The was as described P.L. 1998; PubMed Scopus Google Scholar). are values of two The lines were fitted and values were estimated using the ATP a in is the maximum ATP is and is the values are in of mutations of Asp710 and Asp369 for free ATP binding at of ATP and of lines was done as in The estimated values were for wild type 5 for for and for free energy values are in of mutations of Asp710 and Asn713 for binding of or ATP binding was done as described under “Experimental Procedures.” were fitted to the using the binding of a in which a is the maximum binding and is the The values were for wild type and for a and 1 for D710A a and for a and 10 2 and for a 1 5 1 ATP binding was determined as done Jorgensen P.L. PubMed Scopus Google Scholar). were fitted to the using the ATP binding of a in which a is the maximum ATP binding and is the The values were for wild type and 5 for a 1 5 for D710A and 1 for a and 5 and for a and 3 of E1 → E2 of Na,K-ATPase with cation The for and of E2 or Tl+ or bound or and from with (2Jorgensen P.L. Andersen J.P. J. Membr. Biol. 1988; 103: 95-120Crossref PubMed Scopus (269) Google Scholar). The high affinity to the E2P also to the other but with reduced this segment was to in ATP binding on of with of M. PubMed Scopus Google Scholar) or with ATP at Asp710 or Asp714 1987; PubMed Scopus Google Scholar). J. Biol. Chem. Full Text PDF PubMed Google Scholar) that and the are cells for not in ouabain The → mutation phosphorylated to of wild type whereas → was expressed catalytic activity J. Biol. Chem. Full Text PDF PubMed Google Scholar). were for the residues in Ca-ATPase J. Biol. Chem. Full Text PDF PubMed Google Scholar). role in was on the of binding J. Biochem. PubMed Scopus Google Scholar) and mutation of Asp710 in the α-subunit of renal Na,K-ATPase not with free ATP binding P.L. PubMed Scopus Google Scholar). of Asp710 and Asp714 to coordination of Mg2+ was also in at the active site of cation on the Biochem. J. PubMed Scopus Google Scholar). In mutation of in the sequence Mg2+ affinity about whereas mutation of not with Mg2+ binding J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). For structure-function relationships of the708TGDGVNDSPALKK720 segment in is a that mutations in this segment the protein C. Biol. 1998; PubMed Scopus Google Scholar) and in yeast in Scholar). and with reduced during these mutations expressed in for of high affinity binding of free ATP at and with Mg2+ Jorgensen P.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the present work mutations were in the segment, and 11 of these mutations were expressed in yeast in for of binding of Asp714 were of catalytic of Asp710 and Asn713 reduced Na,K-ATPase activity with ATP were to of Mg2+ for of the transfer of γ-phosphate from ATP to the Mg·E1P[3Na] in the presence of oligomycin and for of the or with and were to for changes in cation binding or in conformational The that Asp710 and Asn713 are important for coordination of Mg2+ in but transition to E2P involves a shift of Mg2+ coordination away from Asp710 and In the E2P the two residues are more important for hydrolysis of the acyl phosphate bond at Asp369. of Asp714 were to PubMed Scopus Google Scholar). The chain were with and Jorgensen P.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The mutations were in Scholar). The sequence of all chain was cells were to PubMed Scopus Google Scholar). yeast cells were in with and to a the of yeast cells wild type Na,K-ATPase at and protein with was as Jorgensen P.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells all other mutations were at °C all was to °C for and with in Scholar). cells were of of membranes at of and protein were as described Jorgensen P.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Assay at 3 mm was as Jorgensen P.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Assay at ATP, mm and 2 mm K+ were done in a as described P.L. Andersen J.P. PubMed Scopus Google Scholar). The binding of these was done as described Jorgensen P.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Jorgensen P.L. PubMed Scopus Google Scholar). each of ATP, specific binding was estimated as the binding in that in In of specific ATP or ouabain binding concentration, lines were fitted using to the ATP binding of in is the maximum ATP and is the were done as described P.L. 1998; PubMed Scopus Google Scholar). Free Mg2+ was calculated using the Bad Chelator program (23Marks P.W. Maxfield F.R. Anal. Biochem. 1991; 193: 61-71Crossref PubMed Scopus (92) Google Scholar). It is from that was for all mutations for Thr708 → Ser and of Asp714 for or The ouabain binding capacities were of protein and in the range as the concentrations of α-subunit protein as estimated from on for mutations of For mutations of Asp714 to or the were expressed to of of but were of all the catalytic that been in this of catalytic activity with of the Asp714 → mutation in Na,K-ATPase and mutations of the residue in Ca-ATPase of J. Biol. Chem. Full Text PDF PubMed Google J. Biol. Chem. Full Text PDF PubMed Google activity and ouabain binding of Ser and mm were done as described under “Experimental and the are values of two or The capacities and of ouabain binding are values for or more are estimated of binding at a of [3H]ouabain concentrations as in in a Na,K-ATPase were done as described under “Experimental and the are values of two or The capacities and of ouabain binding are values for or more are estimated of binding at a of [3H]ouabain concentrations as in in that the for ouabain in vanadate of mutations of Asn713, and were nm and to the for wild type of Asp710 of the affinity for to in Asp710 → and to in the Asp710 → Ala mutation and is the of ouabain is to a in conformational or to with binding of that are required for high affinity ouabain It is from that Na,K-ATPase were severely reduced in mutations of Asp710, Asn713, and at high and ATP concentrations in the In the at 3 mm ATP were reduced or whereas the at ATP were reduced for mutations of and Assay at ATP was to and the of The ATP binding that the affinity of Na,K-ATPase for binding of ATP is that of the in the yeast membranes binding in K+ not The of Na,K-ATPase activity in the at ATP and K+ was more in wild in the at high ATP and K+ of in and shows that the capacities for binding of ATP at were to of ouabain binding for the mutations in the conserved The for ATP binding in the mutations of and were not different from wild type of capacity and a of the affinity for ATP were for the → mutation and for ATP binding of Ser capacities and of ATP binding are values for two to are estimated of binding at a of concentrations as in 2 and in a The capacities and of ATP binding are values for two to are estimated of binding at a of concentrations as in 2 and of Asp710 for or Ala the affinity for ATP 2- to 2 and to in free energy of ATP binding of to of between Asp710 and the negatively charged γ-phosphate of ATP, but the is to the of free energy of ATP binding of the phosphorylated residue Ala Jorgensen P.L. PubMed Scopus Google Scholar). these changes of affinity for ATP on interaction between the two binding of free ATP at was also determined for the mutations of Asp369 → Ala with Asp710 → or Asp710 → The binding in and that these mutations high for ATP with values as as energy and in H. and New Scholar) of the that interaction between the two in the E1 to the between the and phosphate of and mutations of and on ATP binding and as estimated from of ATP of from in 5 and ATP values in 2 and 3 under the that is the mutations P.L. Andersen J.P. PubMed Scopus Google Scholar) using the ATP The ATP values were to values as and in H. and New free energy to ATP binding of between Asp369 and Asp710 was calculated 2 in a of from in 5 and ATP values in 2 and 3 under the that is the mutations P.L. Andersen J.P. PubMed Scopus Google Scholar) using the ATP The ATP values were to values as and in H. and New free energy to ATP binding of between Asp369 and Asp710 was calculated 2 of high affinity binding of free ATP of phosphorylation in mutations of Asn713 in the presence of in the to the Mg·E1P[3Na] and transition to the E2P for Mg2+ for of transfer of γ-phosphate to phosphorylation was determined at of mm that in mm in the presence of mm the of specific phosphorylation free Mg2+ in Mg2+ affinity for wild type 5 was to a of for renal Na,K-ATPase De Pont J.J.H.H.M. The on The of Scholar). of Mg2+ affinity to was for the Asp710 → For the Asp710 → Ala phosphorylation was with Mg2+ not specific phosphorylation was in the range of mm Mg2+ but of the ouabain binding capacity of Mg2+ to 10 mm not phosphorylation of the for ouabain binding for Asp710 → more of was of the of Asn713 The that the of Asp710 is important for Mg2+ binding and phosphorylation and that Asn713 is involved in these processes. the of the ATP for mutations of Asp710 and Asn713, the for K+ of ATP binding in were to the with values about that for wild type for mutations of Asp710 and for mutations of The mutation of Asn713 not Tl+ whereas a of Tl+ affinity was for mutations of Asp710, to with stabilization of the cation sites in the E2 of the for the E1 → E2 conformational from the as described Jorgensen P.L. PubMed Scopus Google Scholar) shows that the mutations of Asp710 or Asn713 a 2- to of with wild type to a shift of conformational the E2 the of the was that of the of Asp369 and Jorgensen P.L. PubMed Scopus Google but the shows also that the of Asp710 and Asn713 are important for stabilization of the E1 Jorgensen P.L. PubMed Scopus Google Scholar). of the mutations on Mg2+ dependence of ouabain binding are in shows the values from the and the that are estimated to the of ouabain The of wild type was in vanadate in phosphate The Asp710 → Ala mutation a of the affinity for Mg2+ in whereas was a of the affinity for Mg2+ in vanadate of mutations of and for binding in ouabain with phosphate or binding was determined as in and the values of the are with the of in Mg2+ dependence of ouabain binding in phosphate was for and of Asp710 and Asp369. of from Mg2+ were estimated using the under the the free energy to Mg2+ binding of between Asp369 and Asp710 was estimated as in 3 to in a binding was determined as in and the values of the are with the of in Mg2+ dependence of ouabain binding in phosphate was for and of Asp710 and Asp369. of from Mg2+ were estimated using the under the the free energy to Mg2+ binding of between Asp369 and Asp710 was estimated as in 3 to on the values in a energy and in H. and New Scholar) was to between Asp710 and Asp369 with to the binding of Mg2+ in the E2 of one of the two of Asp369 or Asp710 of the free energy of binding of whereas mutation of the a The estimated values that the interaction between Asp369 and Asp710 to high affinity binding of Mg2+ in the other Asp710 or Asn713 for the high affinity binding of Mg2+ in the E2 The transition from to E2P involves a shift of Mg2+ coordination away from Asp710 and Asn713 to hydrolysis of the bond at Asp369. in that the two mutations a on the affinity for ouabain binding in phosphate or in of the of a of the for ouabain binding in vanadate, whereas was a in phosphate of the of Asn713 a for ouabain binding in whereas for ouabain in vanadate at wild type of and for affinity for binding of phosphate and 5 binding was determined as in and the values of the are with the of in and values were determined in and Pi or were estimated using the under in a binding was determined as in and the values of the are with the of in and values were determined in and Pi or were estimated using the under The of ouabain binding as a of phosphate or vanadate is in shows values from the and the for phosphate or vanadate Pi or as estimated to the in the The estimated phosphate affinity for wild type is Pi but is to values phosphorylation (2Jorgensen P.L. Andersen J.P. J. Membr. Biol. 1988; 103: 95-120Crossref PubMed Scopus (269) Google Scholar). The Asp710 → Ala mutation the vanadate dependence of ouabain In phosphate and vanadate the Asp710 → Ala mutation the of ouabain binding in Mg2+ and the was whereas the estimated Pi was reduced 2- to The Asn713 → Ala mutation also the of ouabain binding in Mg2+ The that Asp710 is important for the binding of vanadate, whereas Asn713 is more important for phosphorylation from phosphate of the E2P with In the Asp710contributes to coordination of Mg2+ during transfer of γ-phosphate to Asp369 of the Mg·E1P[3Na] with and Asn713 is involved in these processes. Asp710 and Asn713 do not contribute to Mg2+ binding in the E2P with The specific of mutations of Asp710 and Asn713 on Mg2+ affinity of the that transition from to E2P in with is a shift of Mg2+ binding away from Asp710 and In the E2 Asp710 and Asn713 are more important for vanadate binding and for of Asp710 interaction with vanadate and the Asn713 → Ala mutation interferes with phosphorylation of the E2 with with transmission of structural changes that Asp710 is important for the transmission of K+ stimulation of hydrolysis from to the P domain. catalytic are and not to of the protein in and ouabain binding that the α-subunit protein of the mutations is expressed at to wild The of high affinity binding of ATP or also to that the with binding of Mg2+ in and with vanadate or phosphate binding in The large in free energy of ATP binding of the of Asp369 is to of with the γ-phosphate of ATP Jorgensen P.L. PubMed Scopus Google Scholar). The 2- to of ATP affinity of the of Asp710 to a free energy of and is for interaction between the two negatively charged In agreement with this the residues in Ca-ATPase are in the negatively charged surface of the P domain (6Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar). a the γ-phosphate of the bound ATP the negatively charged surface of the P domain in the α-subunit of of the between the domain and the P domain in the of the The two are widely in the structure of Ca-ATPase with two bound (6Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar). Ca-ATPase of not free ATP but with affinity J.P. Jorgensen P.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In of the and of mutations on the for the the is that Asp710 and Asn713 contribute to coordination of Mg2+ in is in agreement with the of the loop in to the phosphorylated Asp369 residue in the high resolution structure of Ca-ATPase (6Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar). The role of Asp710 to Mg2+ in a that of Asp369 to the acyl phosphate Asp369 is the phosphate and Mg2+ to Asp710 in the TGDGVND and Mg2+ the of the phosphate and the two Biochem. J. PubMed Scopus Google Scholar). that of Asp710 or Asn713 for the Ala a shift of the conformational the E2 in with the that of Asp369 for Ala a transition from the E1 to the E2 Jorgensen P.L. PubMed Scopus Google Scholar). The of these negatively charged Mg2+ important for of the → E2P The structural the → E2P transition is to the of from the binding sites of Mg·E1P[3Na] to the Scholar). It important of this transition to shift Mg2+ coordination away from Asp710 and The mutations of the affinity for Mg2+ in the E2·ouabain complex, and shows that of between Asp710 and contribute to binding of Mg2+ in the E2P with on binding has been that one Mg2+ is in the and that Mg2+ is in the J. 1988; PubMed Scopus Google Scopus Google Scholar). The present do not contribute to residues Mg2+ in the E2P but from the segment of the cytoplasmic loop between and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is that Mg2+ to of the M. PubMed Scopus Google which the between the domain and the P domain in Ca-ATPase (6Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar). shift of Mg2+ coordination to the segment of in the domain a to the between the P and with the of or of the segment transition to the E2P (2Jorgensen P.L. Andersen J.P. J. Membr. Biol. 1988; 103: 95-120Crossref PubMed Scopus (269) Google Scholar). of structural changes from the P domain to M4 and between cation residues to from sites that are for with a of to sites that K+ with a of P.L. PubMed Scopus Google Scholar, P.L. 1998; PubMed Scopus Google Scholar). In the E2P that Asp710 and Asn713 are more important for the with vanadate and phosphate and for the of the conformational transition to the cation sites in M5, and P.L. 1998; PubMed Scopus Google Scholar). The of the mutations are and of the of Asp710 vanadate binding and of the of Asn713 the phosphate dependence of ouabain that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. In the the GDGVND segment is important for transmission of the K+ stimulation to hydrolysis of the Asp710 contribute to coordination of vanadate in the transition state J. Biol. Chem. Full Text PDF PubMed Google Scholar). In to vanadate the Asp710 → Ala mutation for but for the in affinity for is of the estimated affinity for phosphate For wild type and the for phosphate are in the in the E2P·ouabain with the that from the ouabain of the to of Google Scholar). The of the Asp710 → Ala mutation on the affinity for ouabain is of the as that of vanadate from wild type whereas the affinity of the Asp369 → Ala mutation for ouabain in Mg2+ is Jorgensen P.L. PubMed Scopus Google Scholar). The Asp710 → Ala mutation also a of the affinity for binding of Tl+ that of the Asp710 interferes with transmission of the structural to the cation sites and ouabain binding domain at the In the this to a role of Asp710 in transmission of the of phospho-enzyme hydrolysis from to the P domain. of vanadate as a phosphate transition state J. Biol. Chem. Full Text PDF PubMed Google that the in the in the transition state for in the E2 The Mg2+ in vanadate with phosphate that Mg2+ in the transition state are in the is in with the of transition state between and or and M. Scholar, Chem. Scopus Google Scholar). The values for Mg2+ in the Asp369 → Ala and mutations in the of vanadate or phosphate are of the as for wild type and mutation of Asp710 in the presence of vanadate The Asp710 → Ala mutation a structure the Mg2+ site that in the transition Asp369 → Ala has been not to with vanadate or phosphate Jorgensen P.L. PubMed Scopus Google Scholar). The present are of the sequence is preserved evolution of cation pumps from to of cation P-type the of the binding protein of a and transport for K+ and Mg2+ (5Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (735) Google Scholar). and for of ouabain to Na,K-ATPase in the presence of vanadate or phosphate described the following of and the to the following for is the required to ouabain is the for and is the ouabain in the binding for vanadate or phosphate in the presence of calculated are the vanadate or phosphate concentrations required to ouabain