Abstract
The central shaft of the catalytic core of ATP synthase, the γ subunit consists of a coiled-coil structure of N- and C-terminal α-helices, and a globular domain. The γ subunit of cyanobacterial and chloroplast ATP synthase has a unique 30-40-amino acid insertion within the globular domain. We recently prepared the insertion-removed α(3)β(3)γ complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central α-helices in γ. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central α-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ε inhibition. In contrast, the change in activity caused by removal of the γ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the γ subunit indirectly regulates complex activity by changing both ADP inhibition and ε inhibition.
Highlights
A conformational change of the ␥ subunit of ATP synthase may be critical for enzyme regulation
The insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition
ATP synthase is a very unique enzyme because the reaction catalyzed at the catalytic sites located on the  subunit is coupled to rotation of the central axis ␥ subunit [10]
Summary
A conformational change of the ␥ subunit of ATP synthase may be critical for enzyme regulation. We postulated that removal of the insertion affects the activity via a conformational change of two central ␣-helices in ␥. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central ␣-helices to each other by way of a disulfide bond formation. The change in activity caused by removal of the ␥ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the ␥ subunit indirectly regulates complex activity by changing both ADP inhibition and ⑀ inhibition. Single molecule analysis of the catalytic turnover of this enzyme indicates that ADP inhibition can be observed as
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