Role of αPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase
Role of αPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase
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DOI：
10.1016/j.abb.2008.01.013
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发表时间：
2008-03-15
影响因子：
3.9
通讯作者：
Ahmad, Zulfiqar
中科院分区：
文献类型：
--
作者：
Brudecki, Laura E.;Grindstaff, Johnny J.;Ahmad, Zulfiqar
研究了α-Phe-291残基在大肠杆菌F1 F0-ATP合酶与磷酸盐结合中的作用。牛线粒体酶的X-射线结构表明，该残基与保守的β R246残基非常接近。在此，我们发现大肠杆菌中的突变α F291 D和α F291 E。coli使F1 F0膜的ATP酶活性降低350倍。然而，保留了显著的氧化磷酸化活性。与野生型相比，MgADP-叠氮化物、MgADP-氟铝酸盐或MgADP-氟钪不抑制突变体的ATP酶活性。然而，7-氯-4-硝基苯并-2-氧杂-1，3-二唑（NBD-Cl）基本上完全抑制野生型ATP酶，突变体中的ATP酶最大抑制率接近75%，尽管反应仍发生在磷酸盐结合口袋中接近α Phe-291的残基β Tyr-297处。抑制特性支持的结论，NBD-Cl反应在PE（空）催化位点，如先前所示的X-射线结构分析。磷酸盐保护野生型NBD-Cl抑制，但在突变体中没有。此外，我们的数据表明，在ATP水解或合成过程中，α-Phe-291与磷酸盐的相互作用可能是不同的。(C)2008年爱思唯尔公司All rights reserved.
The role of alpha Phe-291 residue in phosphate binding by Escherichia coli F1F0-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved beta R246 residue. Herein, we show that mutations alpha F291D and alpha F291E in E. coli reduce the ATPase activity of F1F0 membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by similar to 75%, although reaction still occurred at residue beta Tyr-297, proximal to alpha Phe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in PE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of alpha Phe-291 with phosphate during ATP hydrolysis or synthesis may be distinct. (C) 2008 Elsevier Inc. All rights reserved.