An Insight into the Mechanism of Inhibition and Reactivation of the F1-ATPases by Tentoxin
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- 作者：Jé ; rô ; me Santolini ; Claire Minoletti ; Jean-Marie Gomis ; Claude Sigalat ; Franç ; ois André ; and Francis Haraux
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：May 14, 2002
- 年：2002
- 卷：41
- 期：19
- 页码：6008 - 6018
- 全文大小：286K
- 年卷期：v.41,no.19(May 14, 2002)
- ISSN：1520-4995
文摘
The mechanism of inhibition and reactivation of chloroplast ATP-synthase by the fungalcyclotetrapeptide tentoxin was investigated by photolabeling experiments, binding studies, and kineticanalysis using synthetic analogues of tentoxin. The 
-subunit of chloroplast F1-ATPase (CF1) wasspecifically labeled by a photoactivatable tentoxin derivative, providing the first direct evidence of tentoxinbinding to the -subunit, and 3D homology modeling was used to locate tentoxin in its putative bindingsite at the /
interface. The nonphotosynthetic F1-ATPase from thermophilic bacterium (TF1) proved tobe also tentoxin-sensitive, and enzyme turnover dramatically increased the rate of tentoxin binding to itsinhibitory site, contrary to what was previously observed with 
-depleted CF1 [Santolini, J., Haraux, F.,Sigalat, C., Moal, G., and André, F. (1999) J. Biol. Chem. 274, 849-858]. We propose that tentoxinpreferentially binds to an ADP-loaded pair, and mechanically blocks the catalytic cycle, perhaps bythe impossibility of converting this pair into an ATP-loaded pair. Using 14C-tentoxin and selectedsynthetic analogues, we found that toxin binding to the tight inhibitory site of CF1 exerts some cooperativeeffect on the loose reactivatory site, but that no reciprocal effect exists. When the two tentoxin-bindingsites are filled in reactivated F1-ATPase, they do not exchange their role during catalytic turnover, indicatingan impairment between nucleotide occupancy and the shape of tentoxin-binding pocket. This analysisprovides a mechanical interpretation of the inhibition of F1-ATPase by tentoxin and a clue for understandingthe reactivation process.
-subunit of chloroplast F1-ATPase (CF1) wasspecifically labeled by a photoactivatable tentoxin derivative, providing the first direct evidence of tentoxinbinding to the -subunit, and 3D homology modeling was used to locate tentoxin in its putative bindingsite at the / interface. The nonphotosynthetic F1-ATPase from thermophilic bacterium (TF1) proved tobe also tentoxin-sensitive, and enzyme turnover dramatically increased the rate of tentoxin binding to itsinhibitory site, contrary to what was previously observed with -depleted CF1 [Santolini, J., Haraux, F.,Sigalat, C., Moal, G., and André, F. (1999) J. Biol. Chem. 274, 849-858]. We propose that tentoxinpreferentially binds to an ADP-loaded pair, and mechanically blocks the catalytic cycle, perhaps bythe impossibility of converting this pair into an ATP-loaded pair. Using 14C-tentoxin and selectedsynthetic analogues, we found that toxin binding to the tight inhibitory site of CF1 exerts some cooperativeeffect on the loose reactivatory site, but that no reciprocal effect exists. When the two tentoxin-bindingsites are filled in reactivated F1-ATPase, they do not exchange their role during catalytic turnover, indicatingan impairment between nucleotide occupancy and the shape of tentoxin-binding pocket. This analysisprovides a mechanical interpretation of the inhibition of F1-ATPase by tentoxin and a clue for understandingthe reactivation process.