Consequences of mutations to the phosphorylation site of the α-subunit of Na,K-ATPase for ATP binding and E₁-E₂ conformational equilibrium

TY - JOUR

T1 - Consequences of mutations to the phosphorylation site of the α-subunit of Na,K-ATPase for ATP binding and E1-E2 conformational equilibrium

AU - Pedersen, Per Amstrup

AU - Rasmussen, Jakob H.

AU - Jørgensen, Peter L.

PY - 1996/12/17

Y1 - 1996/12/17

N2 - Expression of Na,K-ATPase in yeast allowed targeting of αβ-units with lethal substitutions at the phosphorylation site αl(D369N)β1 and αl(D369A)/3β1 at the cell surface at the same concentration of α-subunit and [3H]ouabain binding sites as for wild type Na,K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na,K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na,K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the γ-phosphate to the free energy of ATP binding. Analysis of the K+-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the α-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H]ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD= 133 nM for wild type, and [3H]ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the γ-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.

AB - Expression of Na,K-ATPase in yeast allowed targeting of αβ-units with lethal substitutions at the phosphorylation site αl(D369N)β1 and αl(D369A)/3β1 at the cell surface at the same concentration of α-subunit and [3H]ouabain binding sites as for wild type Na,K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na,K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na,K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the γ-phosphate to the free energy of ATP binding. Analysis of the K+-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the α-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H]ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD= 133 nM for wild type, and [3H]ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the γ-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.

UR - http://www.scopus.com/inward/record.url?scp=0030456587&partnerID=8YFLogxK

M3 - Journal article

C2 - 8973179

AN - SCOPUS:0030456587

SN - 0006-2960

VL - 35

SP - 16085

EP - 16093

JO - Biochemistry

JF - Biochemistry

IS - 50

ER -