H^＋—ATPase单位点水解过程中Fo构象的变化

CONFORMATIIONAL CHANGE OF Fo-MOIETY DURING H￣+ -ATPase UNISITE (ATP) HYDROLYSIS

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光，观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时，Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ），也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ，这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ；用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。

For a better understanding of the precise mechanism of nucleotides binding in F1 - moiety to induce conformational change of Fo - moiety, it is necessary to study the conformational interaction between F, and Fo in H+ -ATPase. The intrinsic tryptophan fluorescnce intensity and the quenching constant by Hypocrellin B(HB) are measure to monitor conformational change of Fo resultinng from unisite ATP hydrolysis by F1.The results can be summarized as follows:(1) During unisite ATP hydrolysis of F1 driven by Mg2+, the dynamic change of Fo conformation monitored by tryptophan fluorescence intensity is significant, but no fluorescence intensity change is obsereved in the controls, i. e. addinng ADP instead of the substrate ATP or no substrate was added.(2) The steady-state fluorescence quenching constant Ksv of ATP binding is different from ADP binding in catalytic site of F1 when enzyme: nucleotide= 1: 1. When ATP hydrolysis was driven by Ma2+ or Mn2+ the Ksv changed from state of ATP binding to ADP binding in Fo. This result indicates that after ADP is produced from ATP hydrolysis at FI catalytic site, the conformation of Fo also changes to ADP binding state.(3) In order to further identify the conformational change of Fo-moiety in unisite state of F1(enzyme: ATP or ADP = 1: 1) in the absence of Ma2+, the time resolved fluorescence was measured. The Ksv and average lifetime for ATP binding are 0. 10 and 3. 57ns and for ADP binding 0. 078 and 3. 44 us. The results are consistent with that from the steady - state observation.The above results imply that information of ATP hydrolysis by F1 and of various nucleotides binding to F1(at the tightly nucleotide binding site) can be transferred to Fo - moiety thus results in the conformational change of Fo, so that its function can be controlled.