One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar
One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar
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DOI：
10.1529/biophysj.104.054668
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发表时间：
2005-03-01
影响因子：
3.4
通讯作者：
Kinosita, K
中科院分区：
文献类型：
--
作者：
Sakaki, N;Shimo-Kon, R;Kinosita, K
F-1-ATP酶是一种旋转分子马达，其中中心γ亚基在由α（3）β（3）亚基组成的圆柱体内旋转。旋转是由β亚基上三个催化位点的ATP水解驱动的。在旋转过程中，三个催化位点中有多少被核苷酸填充是一个重要但尚未解决的问题。在这里，我们询问F-1是否在极低的ATP浓度下旋转，预计位点占用率很低。我们在光学显微镜下观察到在γ亚基上携带珠双链体的单个F1分子的旋转。时间平均旋转速率与ATP浓度成正比，直到200 pM，ATP结合的表观速率常数为2 x 10（7）M-1 s（-1）。类似的速率常数表征了溶液中的大量ATP水解，其在6 mM和60 nM ATP之间遵循简单的Michaelis-Menten方案。F-1产生了类似于40 pN的相同扭矩。在2 mM、60 nM和2 nM ATP下，这些结果指出，一个旋转机制，管理整个范围内的纳摩尔到毫摩尔ATP，虽然两种机制之间的竞争不能被解雇。低于1 nM ATP时，我们观察到较少的规则旋转，表明出现了另一种反应方案。
F-1-ATPase is a rotary molecular motor in which the central gamma-subunit rotates inside a cylinder made of alpha(3)beta(3)-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the beta-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F-1 rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F1 molecules that carried a bead duplex on the gamma-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 x 10(7) M-1 s(-1). A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F-1 produced the same torque of similar to40 pN . nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.