Santolini Jérôme, Minoletti Claire, Gomis Jean-Marie, Sigalat Claude, André François, Haraux Francis
Service de Bioénergétique, DBJC, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France.
Biochemistry. 2002 May 14;41(19):6008-18. doi: 10.1021/bi015938z.
The mechanism of inhibition and reactivation of chloroplast ATP-synthase by the fungal cyclotetrapeptide tentoxin was investigated by photolabeling experiments, binding studies, and kinetic analysis using synthetic analogues of tentoxin. The alpha-subunit of chloroplast F(1)-ATPase (CF(1)) was specifically labeled by a photoactivatable tentoxin derivative, providing the first direct evidence of tentoxin binding to the alpha-subunit, and 3D homology modeling was used to locate tentoxin in its putative binding site at the alpha/beta interface. The non-photosynthetic F(1)-ATPase from thermophilic bacterium (TF(1)) proved to be also tentoxin-sensitive, and enzyme turnover dramatically increased the rate of tentoxin binding to its inhibitory site, contrary to what was previously observed with epsilon-depleted CF(1) [Santolini, J., Haraux, F., Sigalat, C., Moal, G., and André, F. (1999) J. Biol. Chem. 274, 849-858]. We propose that tentoxin preferentially binds to an ADP-loaded alpha beta pair, and mechanically blocks the catalytic cycle, perhaps by the impossibility of converting this alpha beta pair into an ATP-loaded alpha beta pair. Using (14)C-tentoxin and selected synthetic analogues, we found that toxin binding to the tight inhibitory site of CF(1) exerts some cooperative effect on the loose reactivatory site, but that no reciprocal effect exists. When the two tentoxin-binding sites are filled in reactivated F(1)-ATPase, they do not exchange their role during catalytic turnover, indicating an impairment between nucleotide occupancy and the shape of tentoxin-binding pocket. This analysis provides a mechanical interpretation of the inhibition of F(1)-ATPase by tentoxin and a clue for understanding the reactivation process.
通过光标记实验、结合研究以及使用滕毒素合成类似物的动力学分析，研究了真菌环四肽滕毒素对叶绿体ATP合酶的抑制和再激活机制。叶绿体F₁ - ATP酶（CF₁）的α亚基被一种可光活化的滕毒素衍生物特异性标记，这为滕毒素与α亚基结合提供了首个直接证据，并且利用三维同源建模将滕毒素定位在其位于α/β界面的假定结合位点。嗜热细菌的非光合F₁ - ATP酶（TF₁）也被证明对滕毒素敏感，并且酶的周转显著增加了滕毒素与其抑制位点的结合速率，这与之前在ε缺失的CF₁中观察到的情况相反[Santolini, J., Haraux, F., Sigalat, C., Moal, G., and André, F. (1999) J. Biol. Chem. 274, 849 - 858]。我们提出滕毒素优先结合到负载ADP的αβ二聚体上，并通过机械方式阻断催化循环，可能是由于无法将这个αβ二聚体转化为负载ATP的αβ二聚体。使用¹⁴C - 滕毒素和选定的合成类似物，我们发现毒素与CF₁紧密抑制位点的结合对松散的再激活位点产生了一些协同效应，但不存在反向效应。当在再激活的F₁ - ATP酶中两个滕毒素结合位点都被占据时，它们在催化周转过程中不会交换其作用，这表明核苷酸占据情况与滕毒素结合口袋形状之间存在损伤。该分析为滕毒素对F₁ - ATP酶的抑制提供了一种机理解释，并为理解再激活过程提供了线索。
