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首页> 外文期刊>Biochemistry >Consequences of Mutations to the Phosphorylation Site of the α-Subunit of Na,K-ATPase for ATP Binding and E_1-E_2 Conform ational Equilibrium
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Consequences of Mutations to the Phosphorylation Site of the α-Subunit of Na,K-ATPase for ATP Binding and E_1-E_2 Conform ational Equilibrium

机译：Na，K-ATPase的α亚基的磷酸化位点突变对ATP结合和E_1-E_2构象平衡的影响

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Expression of Na,K-ATPase in yeast allowed targeting of αβ-units with lethal substitutions at the phosphorylation site α1(D369N)β1 and α1(D369A)β1 at the cell surface at the same concentration of a-subunit and [~3H]ouabain binding sites as forwild 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 (Kb =2.8 nM) that was 39-fold higher than for wild type Na,K-ATPase (K_D = 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 hydtophobic substitution at Asp~(369) of the α-subunit caused a large shift in conformational equilibrium toward the E_2-form. This was accompanied by a large increase in affinity for [~3H]ouabain in Mg~(2+) medium with K_D = 4.9 nM for D369A compared to Kb = 51 nM for D369N and K_D = 133 nM for wild type, and [~3H]ouabain binding (K_D = 153 nM) to D369A was detectable even in absence of Mg~(2+). In addition to its function as receptor of the γ-phosphate of ATP, Asp~(369) has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E_1 -- E2 transitions which are coupled to reorientation of cation sites and changesin affinity for digitalis glycosides.
机译：酵母中Na，K-ATPase的表达允许以相同浓度的a-亚基和[〜3H]在细胞表面磷酸化位点α1（D369N）β1和α1（D369A）β1处具有致命取代的αβ-单元靶向。废除了哇巴因结合位点，如野生型Na，K-ATPase，磷酸化和与钒酸盐的反应，并且该突变没有Na，K-ATPase或K-磷酸酶活性。 [〜3H] -ATP的平衡结合揭示了D369A突变对ATP的内在高亲和力（Kb = 2.8 nM），比野生型Na，K-ATPase（K_D = 109 nM）高39倍。对ADP的亲和力未受影响，表明残基369处的负电荷决定了γ-磷酸盐对ATP结合自由能的贡献。对K〜+ -ATP拮抗作用的分析表明，电荷的减少和疏水取代在α-亚基的Asp〜（369）处，构象平衡向E_2-型发生了很大的变化。这伴随着对Mg〜（2+）培养基中[〜3H] ouabain的亲和力大大增加，D369A的K_D = 4.9 nM，而D369N的Kb = 51 nM和野生型的K_D = 133 nM。即使没有Mg〜（2+），也可以检测到3H]哇巴因与D369A的结合（K_D = 153 nM）。 Asp〜（369）除具有ATP的γ-磷酸受体功能外，在调节对ATP的亲和力方面还具有重要的短程催化功能，并在控制E_1-E2跃迁方面具有长程功能，阳离子位点的重新定位和对洋地黄苷的亲和力变化。

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