Brudecki Laura E, Grindstaff Johnny J, Ahmad Zulfiqar
Department of Biological Sciences, Box 70703, East Tennessee State University, Johnson City, TN 37614, USA.
Arch Biochem Biophys. 2008 Mar 15;471(2):168-75. doi: 10.1016/j.abb.2008.01.013. Epub 2008 Jan 26.
The role of alphaPhe-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 betaR246 residue. Herein, we show that mutations alphaF291D and alphaF291E 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 approximately 75%, although reaction still occurred at residue betaTyr-297, proximal to alphaPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in betaE (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 alphaPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.
研究了大肠杆菌F1F0 - ATP合酶中αPhe - 291残基在磷酸结合中的作用。牛线粒体酶的X射线结构表明，该残基与保守的βR246残基位置相近。在此，我们表明大肠杆菌中αF291D和αF291E突变使F1F0膜的ATP酶活性降低350倍。然而，仍保留显著的氧化磷酸化活性。与野生型不同，突变体的ATP酶活性不受MgADP - 叠氮化物、MgADP - 氟铝酸盐或MgADP - 氟钪的抑制。而7 - 氯 - 4 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂茂（NBD - Cl）基本完全抑制野生型ATP酶，突变体中的ATP酶最大抑制约75%，尽管在磷酸结合口袋中靠近αPhe - 291的βTyr - 297残基处仍有反应发生。抑制特性支持了NBD - Cl在βE（空）催化位点发生反应的结论，如先前X射线结构分析所示。磷酸盐可保护野生型免受NBD - Cl抑制，但对突变体无效。此外，我们的数据表明，在ATP水解或合成过程中，αPhe - 291与磷酸盐的相互作用可能不同。
