Modulatory ATP Binding Affinity in Intermediate States of E2P Dephosphorylation of Sarcoplasmic Reticulum Ca2+-ATPase
Modulatory ATP Binding Affinity in Intermediate States of E2P Dephosphorylation of Sarcoplasmic Reticulum Ca2+-ATPase
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DOI：
10.1074/jbc.m110.206094
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发表时间：
2011-04-01
影响因子：
4.8
通讯作者：
Andersen, Jens Peter
中科院分区：
文献类型：
--
作者：
Clausen, Johannes D.;McIntosh, David B.;Andersen, Jens Peter
ATP调节E2 P去磷酸化的肌浆网Ca 2 +-ATP酶野生型和突变体形式的机制进行了研究，在核苷酸结合研究的状态类似的各种中间体的去磷酸化反应，通过结合金属氟化物，钒酸盐，或毒胡萝卜素。野生型Ca 2 +-ATP酶显示出对E2 P基态类似物的ATP亲和力为4 μ M，对E2 P过渡态和产物态类似物的ATP亲和力为1 μ M，对E2脱磷酸酶的ATP亲和力为11 μ M。因此，ATP结合相对于基态稳定过渡态和产物态，从而解释ATP对去磷酸化的加速作用。用丝氨酸取代Phe（487）（N-结构域），用亮氨酸取代Arg（560）（N-结构域），或用丙氨酸或谷氨酸取代Arg（174）（A-结构域）降低所有E2/E2 P中间状态中的ATP亲和力。Ile（188）（A-结构域）的丙氨酸取代增加了ATP亲和力，尽管ATP对去磷酸化的加速被破坏，因此表明Ile（188）在ATP调节中的关键作用是基于机制的，而不是与核苷酸的结合相关。具有Lys（205）（A-结构域）或Glu（439）（N-结构域）的丙氨酸替代的突变体表现出ATP对E2 P去磷酸化的异常抑制，这是由于ATP结合增加了E2 P基态相对于过渡态的稳定性。通过在A-M1接头中插入四个甘氨酸稳定的Ca（2）E2 P的ATP亲和力与E2 P基态的ATP亲和力相似，但该突变体的无Ca 2 + E1状态显示ATP亲和力降低3个数量级。
The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca2+-ATPase displays an ATP affinity of 4 mu M for the E2P ground state analog, 1 mu M for the E2P transition state and product state analogs, and 11 mu M for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe(487) (N-domain) with serine, Arg(560) (N-domain) with leucine, or Arg(174) (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile(188) (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile(188) in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys(205) (A-domain) or Glu(439) (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca(2)E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca2+-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.