期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 286, 期 13, 页码 11792-11802出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110.206094
关键词
-
资金
- Danish Medical Research Council
- Novo Nordisk Foundation
- Danish National Research Foundation (PUMPKIN Centre)
- Centre for Membrane Pumps in Cells and Disease, PUMPKIN, Danish National Research Foundation
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据