| Item type |
学術雑誌論文 / Journal Article_02(1) |
| 公開日 |
2012-05-17 |
| タイトル |
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タイトル |
Stable Structural Analog of Ca^<2+>-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca^<2+> Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding |
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言語 |
en |
| 言語 |
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言語 |
eng |
| 資源タイプ |
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資源タイプ |
journal article |
| 著者 |
大保, 貴嗣
Danko, Stefania
Yamasaki, Kazuo
Suzuki, Hiroshi
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| 書誌情報 |
Journal of Biological Chemistry
巻 285,
号 32,
p. 24538-24547,
発行日 2010-08-01
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| ISSN |
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収録物識別子タイプ |
PISSN |
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収録物識別子 |
0021-9258 |
| DOI |
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識別子タイプ |
DOI |
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関連識別子 |
10.1074/jbc.M110.144535 |
| 抄録 |
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内容記述タイプ |
Abstract |
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内容記述 |
We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca^<2+> at the transport sites (E2PCa_2) of sarcoplasmic reticulum Ca^<2+>-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca^<2+> deocclusion/release to the lumen; E1PCa_2 → E2PCa_2 → E2P + 2Ca^<2+>. Stabilization was achieved by elongation of the Glu^<40>-Ser^<48> loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly^<46>/Lys^<47> and by binding of beryllium fluoride (BeFx) to the phosphorylation site of the Ca^<2+>-bound ATPase (E1Ca_2). The complex E2Ca_2·BeF_3− was also produced by lumenal Ca^<2+> binding to E2·BeF_3− (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25℃. Only BeFx, but not AlFx or MgFx, produced the E2PCa_2 structural analog. Complex formation required binding of Mg^<2+>, Mn^<2+>, or Ca^<2+> at the catalytic Mg^<2+> site. Results reveal that the phosphorylation product E1PCa_2 and the E2P ground state (but not the transition states) become competent to produce the E2PCa_2 transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca^<2+> release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu^<40>-Ser^<48> linker and of Mg^<2+> at the catalytic site in these processes. |
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言語 |
en |
| 注記 |
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内容記述タイプ |
Other |
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注記 |
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. |
| 資源タイプ |
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内容記述タイプ |
Other |
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資源タイプ |
text |
| 著者版フラグ |
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出版タイプ |
VoR |
| フォーマット |
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内容記述タイプ |
Other |
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内容記述 |
application/pdf |
| ID(XooNIps) |
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20529842 |
| 閲覧数(XooNIps) |
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| ダウンロード数(XooNIps) |
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822 |
4540.pdf (1.8 MB)
