Purine but not pyrimidine nucleotides support rotation of F1-ATPase
Purine but not pyrimidine nucleotides support rotation of F1-ATPase
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DOI：
10.1074/jbc.m102200200
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发表时间：
2001-07-06
影响因子：
4.8
通讯作者：
Kinosita, K
中科院分区：
文献类型：
--
作者：
Noji, H;Bald, D;Kinosita, K
F-1-ATP酶的结合变化模型预测其旋转与核苷酸的三个催化位点的亲和力的变化密切相关。如果是这样的话，核苷酸结构的细微差异可能对旋转有明显的影响。在这里，我们通过单分子成像显示嘌呤核苷酸ATP，GTP和ITP支持旋转，但嘧啶核苷酸UTP和CTP不支持旋转，这表明嘌呤中的额外环对于该分子马达的正常操作是不可或缺的。虽然这三种嘌呤核苷酸以不同的速率与酶结合，但都显示出相似的旋转特征：逆时针旋转，每个核苷酸分子水解驱动120度的步骤，偶尔的后退步骤，类似于40皮牛顿（pN）的旋转扭矩。nm，并且在类似于80 pN（.）nm.后一种特征可能是由蛋白质结构中内置的旋转机制决定的，嘌呤核苷酸可以激发该机制。对于ATP和GTP，即使当水解自由能为-80 pN（.）nm/分子，表明类似于100%效率。重组的F0 F1-ATP酶通过水解ATP、GTP和ITP而主动地转移质子，但CTP和UTP甚至不被水解。分离的F-1非常缓慢地水解UTP（但不是CTP），这表明可能从旋转解偶联。
The binding change model for the F-1-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of similar to 40 piconewtons (pN)(.)nm, and mechanical work done in a step of similar to 80 pN(.)nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN(.)nm/molecule, indicating similar to 100% efficiency. Reconstituted F0F1-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F-1 very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.