University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, Rockville, Maryland 20850, USA.
Biochemistry. 2009 Mar 24;48(11):2330-9. doi: 10.1021/bi801921j.
The minichromosome maintenance (MCM) helicase is the replicative helicase in archaea. The enzyme utilizes the energy derived from ATP hydrolysis to translocate along one strand of the DNA and unwind the complementary strand. Here, the effect of DNA and ATP on the thermostability of the Methanothermobacter thermautotrophicus MCM protein was determined by differential scanning calorimetry. The MCM protein shows a single thermal transition at 67 degrees C. The stability is dramatically altered with the appearance of a second thermal transition up to 10 degrees C higher in the presence of DNA and either ATP or ADP-AlF(4)(-), a transition-state analogue of ATP, bound to MCM. In the presence of DNA and ADP or the nonhydrolyzable ATP analogues ATPgammaS and AMP-PNP, however, only a single thermal transition is observed at temperatures slightly higher than the transition temperature of MCM alone. Thus, the results suggest that ATP hydrolysis proceeds through a transition state that decouples an interaction between the N-terminal DNA binding domain and the C-terminal catalytic domain in the presence of DNA.
微小染色体维持（MCM）解旋酶是古菌中的复制解旋酶。该酶利用 ATP 水解产生的能量沿 DNA 的一条链移动，并解开互补链。在这里，通过差示扫描量热法测定了 DNA 和 ATP 对产甲烷菌热自养 MCM 蛋白热稳定性的影响。MCM 蛋白在 67°C 时显示单一热转变。在 DNA 存在下，稳定性会发生明显变化，出现第二个热转变，温度比单独的 MCM 高 10°C，同时结合 MCM 的是 ATP 或 ADP-AlF(4)(-)，一种 ATP 的过渡态类似物。然而，当 MCM 与 DNA 结合，并且存在 ADP 或非水解的 ATP 类似物 ATPgammaS 和 AMP-PNP 时，仅在稍高于 MCM 单独转变温度的温度下观察到单一的热转变。因此，结果表明，在 DNA 存在下，ATP 水解通过一个过渡态进行，该过渡态解耦了 N 端 DNA 结合域和 C 端催化域之间的相互作用。
