Interactions of T273 and E275 with the 
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- 作者：Kathryn W. Boltz and Wayne D. Frasch
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：July 12, 2005
- 年：2005
- 卷：44
- 期：27
- 页码：9497 - 9506
- 全文大小：305K
- 年卷期：v.44,no.27(July 12, 2005)
- ISSN：1520-4995
文摘
In Escherichia coli F1Fo ATP synthase, 
T273 mutants that eliminate the ability to form ahydrogen bond to 
V265 were incapable of ATP synthase-dependent growth and ATPase-dependent protonpumping, had very low rates of ATPase activity catalyzed by purified F1, and had significantly decreasedsensitivity to inhibition by Mg2+-ADP-AlFn species, while T273D and T273N mutants which maintainedor increased the hydrogen bond strength maintained or increased catalytic activity. The P262G mutationthat increases the potential flexibility of the rigid sleeve that surrounds the subunit C-terminus alsovirtually eliminated ATPase activity and susceptibility to Mg2+-ADP-AlFn inhibition. The E275 mutantsthat retained the ability to form the V265 hydrogen bond had higher ATPase activity than those thateliminated the hydrogen bond. These results provide evidence that the ability to form hydrogen bondsbetween V265 and the subunit C-terminus contributes significantly to the rate-limiting step of catalysisand to the ability of the F1Fo ATP synthase to use a proton gradient to drive ATP synthesis. The loss ofactivity observed with P262G may result from increased flexibility conferred by glycine that decreasesthe efficiency of communication between the subunit-V265 hydrogen bonds and the Walker B aspartateat the catalytic site. The partial loss of coupling observed with T273 mutants that eliminate the V265hydrogen bond is consistent with participation of this hydrogen bond in the escapement mechanism forATP synthesis in which interactions between the subunit and (
)3 ring prevent rotation until the emptycatalytic site binds substrate.
T273 mutants that eliminate the ability to form ahydrogen bond to V265 were incapable of ATP synthase-dependent growth and ATPase-dependent protonpumping, had very low rates of ATPase activity catalyzed by purified F1, and had significantly decreasedsensitivity to inhibition by Mg2+-ADP-AlFn species, while T273D and T273N mutants which maintainedor increased the hydrogen bond strength maintained or increased catalytic activity. The P262G mutationthat increases the potential flexibility of the rigid sleeve that surrounds the subunit C-terminus alsovirtually eliminated ATPase activity and susceptibility to Mg2+-ADP-AlFn inhibition. The E275 mutantsthat retained the ability to form the V265 hydrogen bond had higher ATPase activity than those thateliminated the hydrogen bond. These results provide evidence that the ability to form hydrogen bondsbetween V265 and the subunit C-terminus contributes significantly to the rate-limiting step of catalysisand to the ability of the F1Fo ATP synthase to use a proton gradient to drive ATP synthesis. The loss ofactivity observed with P262G may result from increased flexibility conferred by glycine that decreasesthe efficiency of communication between the subunit-V265 hydrogen bonds and the Walker B aspartateat the catalytic site. The partial loss of coupling observed with T273 mutants that eliminate the V265hydrogen bond is consistent with participation of this hydrogen bond in the escapement mechanism forATP synthesis in which interactions between the subunit and ()3 ring prevent rotation until the emptycatalytic site binds substrate.