From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656.
the Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551.
J Biol Chem. 2013 Nov 8;288(45):32700-32707. doi: 10.1074/jbc.M113.506329. Epub 2013 Oct 2.
V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μM ATP (<<Km), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10(6) M(-1) s(-1). At 40 mM ATP (>>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.
V-ATPases 是旋转分子马达，通常作为质子泵发挥作用。我们最近解析了嗜热链球菌 V-ATPase（EhV1）V1 亚基的晶体结构，并提出了其旋转机制模型。在这里，我们使用无负载探针的单分子分析对 EhV1 的旋转动力学进行了表征。EhV1 逆时针旋转，呈现出两种明显的旋转状态，即清晰和不清晰，表明转子和定子之间的相互作用不稳定。我们详细分析了清晰状态以获得动力学参数。旋转速率符合米氏动力学，在 26°C 时最大旋转速率（Vmax）为 107 转/秒，米氏常数（Km）为 154 μM。在所有测试的 ATP 浓度下，EhV1 仅显示三个间隔 120°/转的停顿，并且没有解析出亚步骤，与嗜热脂肪芽孢杆菌 V1-ATPase（TtV1）的情况相同。在 10 μM ATP（<<Km）时，ATP 等待停顿持续时间的分布很好地符合单指数衰减函数。ATP 的二级结合速率常数为 2.3×10(6) M(-1) s(-1)。在 40 mM ATP（>>Km）时，催化停顿持续时间的分布可以通过两个时间常数为 2.6 和 0.5 ms 的连续反应来重现。这些动力学参数与 TtV1 的相似。我们的结果确定了 V1-ATPases 旋转催化的共同特性，这些特性与 F1-ATPases 的特性不同，将进一步加深我们对旋转分子马达的一般机制的理解。
