Fluoroacetate dehalogenases (FAcD), a homodimeric enzyme, catalyzes the conversion of fluoroacetic acid to glycolic acid (GoA). It has been proved that the enzyme has a half-of-the-site reactivity. Namely, its catalytic (C) subunit converts the first substrate to a covalent intermediate; then, the non-catalytic (NC) subunit binds a second substrate and promotes the conversion of the intermediate in the C subunit into the final product. After the release of the product, the C subunit becomes the NC subunit, and the previous NC subunit becomes the C subunit. To elucidate the detailed mechanism behind this cooperative catalysis, we have conducted microsecond-scale MD simulations along the reaction pathway. The simulations indicate that the substrate in the NC subunit induces W185 and Y141 adopting an open conformation in the C subunit. The opening of W185 (C) facilitates the entry of catalytic water, enhancing the catalytic activity for product formation, while the opening of Y141 (C) creates an unfavorable environment for product binding, promoting its release. An interaction network analysis reveals that the substrate in the NC subunit can induce conformational changes through a conserved water chain at the interface.

氟乙酸脱卤酶 (FAcD) 是一种同型二聚体酶，可催化氟乙酸转化为乙醇酸 (GoA)。已证明该酶具有半位点反应活性。即，其催化（C）亚基将第一底物转化为共价中间体；然后，非催化 (NC) 亚基结合第二个底物并促进 C 亚基中的中间体转化为最终产物。产品发布后，C亚基变为NC亚基，之前的NC亚基变为C亚基。为了阐明这种协同催化背后的详细机制，我们沿着反应途径进行了微秒级的MD模拟。模拟表明 NC 亚基中的底物诱导 W185 和 Y141 在 C 亚基中采用开放构象。 W185（C）的开放有利于催化水的进入，增强产物形成的催化活性，而Y141（C）的开放为产物结合创造了不利的环境，促进其释放。相互作用网络分析表明，NC 亚基中的底物可以通过界面处的保守水链诱导构象变化。