TY - JOUR

T1 - Importance of conserved α-subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase

AU - Pedersen, Per Amstrup

AU - Jorgensen, Jesper R.

AU - Jorgensen, Peter Leth

PY - 2000/12/1

Y1 - 2000/12/1

N2 - The segment 708TGDGVNDSPALKK720 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 Asp710 contributes 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 Ash713, 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 Ash713 → Ala interferes with phosphorylation from P(i) 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+ simulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.

AB - The segment 708TGDGVNDSPALKK720 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 Asp710 contributes 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 Ash713, 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 Ash713 → Ala interferes with phosphorylation from P(i) 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+ simulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.

UR - http://www.scopus.com/inward/record.url?scp=0034528692&partnerID=8YFLogxK

U2 - 10.1074/jbc.M005610200

DO - 10.1074/jbc.M005610200

M3 - Journal article

C2 - 10982798

AN - SCOPUS:0034528692

VL - 275

SP - 37588

EP - 37595

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 48

ER -