Insight into the bind-lock mechanism of the yeast mitochondrial ATP synthase inhibitory peptide
Insight into the bind-lock mechanism of the yeast mitochondrial ATP synthase inhibitory peptide
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DOI：
10.1021/bi700522v
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发表时间：
2007-07-24
期刊：
影响因子：
2.9
通讯作者：
Haraux, Francis
中科院分区：
文献类型：
--
作者：
Corvest, Vincent;Sigalat, Claude;Haraux, Francis
研究了酵母线粒体f -1-ATP酶调控肽IF1对其抑制的机制，并将非催化位点用焦磷酸预处理冷冻，模拟ATP填充。这允许确认催化位点占用和IF1结合率之间的不匹配，而没有由于ATP与非催化位点结合缓慢而产生的动力学限制。这些数据加强了先前提出的两步机制，其中IF1松散结合由催化状态决定，IF1锁定依赖于转换并与IF1释放竞争(Corvest， V., Sigalat， C., Venard， R., Falson， P., Mueller， D. M., and Haraux， F. (2005) J. Biol。化学，280,9927 -9936)。他们还证明，非催化位点在其结合中起次要作用，而非催化位点会轻微调节IF1进入酶。研究还表明，IF1与装载mgadp的f -1- atp酶的松散结合非常缓慢，IF1与atp水解的f -1- atp酶的结合在微摩尔范围内严重降低核苷酸结合，在亚毫摩尔范围内适度降低核苷酸结合。综上所述，这些观察结果提出了一个总的抑制过程的轮廓。在第一个催化循环中，IF1与新结合的ATP松散地结合在催化位点上，当ATP在第二个位点水解时，IF1被锁定。在第二个循环中，IF1阻断ATP水解抑制ATP被捕获在第三位点，并且在高ATP浓度下，也抑制ADP从第二位点释放。这个模型也为理解为什么IF1在ATP合成过程中不结合ATP合酶提供了线索。
The mechanism of yeast mitochondrial F-1-ATPase inhibition by its regulatory peptide IF1 was investigated with the noncatalytic sites frozen by pyrophosphate pretreatment that mimics filling by ATP. This allowed for confirmation of the mismatch between catalytic site occupancy and IF1 binding rate without the kinetic restriction due to slow ATP binding to the noncatalytic sites. These data strengthen the previously proposed two-step mechanism, where IF1 loose binding is determined by the catalytic state and IF1 locking is turnover-dependent and competes with IF1 release (Corvest, V., Sigalat, C., Venard, R., Falson, P., Mueller, D. M., and Haraux, F. (2005) J. Biol. Chem. 280, 9927-9936). They also demonstrate that noncatalytic sites, which slightly modulate IF1 access to the enzyme, play a minor role in its binding. It is also shown that loose binding of IF1 to MgADP-loaded F-1-ATPase is very slow and that IF1 binding to ATP-hydrolyzing F-1-ATPase decreases nucleotide binding severely in the micromolar range and moderately in the submillimolar range. Taken together, these observations suggest an outline of the total inhibition process. During the first catalytic cycle, IF1 loosely binds to a catalytic site with newly bound ATP and is locked when ATP is hydrolyzed at a second site. During the second cycle, blocking of ATP hydrolysis by IF1 inhibits ATP from becoming entrapped on the third site and, at high ATP concentrations, also inhibits ADP release from the second site. This model also provides a clue for understanding why IF1 does not bind ATP synthase during ATP synthesis.