Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.
Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.
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双位点激活发生在天然和核苷酸耗尽的线粒体 F1-ATP 酶上。
DOI：
10.1042/bj3301037
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Boyer,PD
中科院分区：
文献类型：
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作者：
Milgrom,YM;Murataliev,MB;Boyer,PD
本文报道了牛心线粒体F_1-ATP酶的单中心催化及由单中心催化向多中心催化转变的实验结果。非常缓慢的单位点ATP水解显示在没有紧密结合的核苷酸存在和缓冲液中有或没有Pi的情况下发生。在不同的固定酶浓度下，随着ATP浓度的增加，向更高速率的过渡和致力于水解的结合ATP的量的测量提供了证据，即第二个位点的填充可以启动接近最大的周转率。它们提供了速率常数信息，并表明第二个位点的表观Km约为2μM，Vmax为10 s-1，如其他人所建议的那样，是不起作用的。仔细的初速度测量也消除了其他不确定的Km值，并与接近最大水解速率的双位点活化一致，aKm约为130μM，Vmax约为700 s-1。然而，结果并没有消除额外的“隐藏”Km值与similarVmax：Km比值的可能性。最近的数据TNP-ATP和ATP之间的竞争揭示了第三个催化位点的ATP在毫摩尔浓度范围内。这一结果和本文报道的结果表明，线粒体F1-ATP酶在双位点催化中可以达到接近最大的活性。我们的数据也增加了证据，最近的索赔，线粒体F1-ATP酶不显示催化位点协同性，是无效的。
Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Piin the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparentKmfor a second site of about 2μM andVmaxof 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggestedKmvalues and are consistent with bi-site activation to near maximal hydrolysis rates, with aKmof about 130μM andVmaxof about 700 s-1. However, the results do not eliminate the possibility of additional ‘hidden’Kmvalues with similarVmax:Kmratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid.