Itoh Hiroyasu, Takahashi Akira, Adachi Kengo, Noji Hiroyuki, Yasuda Ryohei, Yoshida Masasuke, Kinosita Kazuhiko
Tsukuba Research Laboratory, Hamamatsu Photonics KK, Joko, Hamamatsu 431-3103, Japan.
Nature. 2004 Jan 29;427(6973):465-8. doi: 10.1038/nature02212.
ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.
三磷酸腺苷（ATP）是生物体内主要的能量货币，由二磷酸腺苷（ADP）和无机磷酸在能量需求反应中通过ATP合酶合成。ATP合酶的F1部分，也称为F1 - ATP酶，起着旋转分子马达的作用：在体外，其γ亚基相对于周围的α3β3亚基旋转，在β亚基上的三个独立催化位点水解ATP。人们普遍认为，由质子流经ATP合酶相关的F0部分驱动的γ亚基反向旋转，会导致生物系统中ATP的合成。在此，我们提供了机械能驱动ATP化学合成的直接证据。我们将磁珠附着在玻璃表面分离的F1的γ亚基上，并使用电磁铁旋转磁珠。通过荧光素酶 - 荧光素反应检测到，沿适当方向的旋转导致培养基中出现ATP。这表明作用于蛋白质机器上一个特定点的矢量力（扭矩）可以影响在物理上遥远的催化位点发生的化学反应，驱动反应远离平衡状态。
