Bruman Sofya M, Zubareva Valeria M, Shugaeva Tatiana E, Lapashina Anna S, Feniouk Boris A
Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119991, Russia.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia.
Biochemistry (Mosc). 2025 Mar;90(3):374-388. doi: 10.1134/S0006297924602600.
Proton FF ATP synthase catalyzes the formation of ATP from ADP and inorganic phosphate coupled with transmembrane proton transfer using the energy of the protonmotive force (). As decreases, the direction of the reaction is reversed and the enzyme generates , transferring protons across the membrane using the energy of ATP hydrolysis. ATPase activity of the enzyme can be suppressed by ADP in a non-competitive manner (ADP-inhibition), and in a number of bacteria, it can be inhibited by conformational changes in the regulatory C-terminal domain of the ε subunit. Lauryldimethylamine oxide (LDAO), a zwitterionic detergent, is known to attenuate both of these inhibitory mechanisms, significantly increasing the ATPase activity of the enzyme. For this reason, LDAO is sometimes used for semi-quantitative estimation of the enzyme's susceptibility to these regulatory mechanisms. However, the binding site of LDAO in ATP synthase remains unknown. The mechanism by which the detergent counteracts ADP-inhibition and the inhibition involving the ε subunit is also unclear. We performed molecular docking and predicted that LDAO binding might occur at the catalytic site of ATP synthase, whether empty or containing nucleotides. Molecular dynamics simulations showed that LDAO could affect the mobility of the loop in the β subunit (residues β404-415 in ATP synthase) near the catalytic site. Mutagenesis of residue β409 in the enzyme and the corresponding β419 residue in the ATP synthase revealed that the type of side chain of this residue indeed affects LDAO-dependent stimulation of ATPase activity. We also found that LDAO activates the enzyme more strongly in the presence of 100 mM sulfate compared to sulfate-free medium. This phenomenon is likely due to the enhancement of ADP-inhibition of the enzyme by sulfate.
质子FF型ATP合酶利用质子动力势的能量催化由ADP和无机磷酸形成ATP，并偶联跨膜质子转移。随着质子动力势降低，反应方向逆转，该酶利用ATP水解的能量产生质子动力势，将质子跨膜转运。该酶的ATP酶活性可被ADP以非竞争性方式抑制（ADP抑制），并且在许多细菌中，它可被ε亚基的调节性C末端结构域的构象变化抑制。月桂基二甲基氧化胺（LDAO），一种两性离子去污剂，已知可减弱这两种抑制机制，显著增加该酶的ATP酶活性。因此，LDAO有时用于半定量评估该酶对这些调节机制的敏感性。然而，LDAO在ATP合酶中的结合位点仍然未知。去污剂抵消ADP抑制和涉及ε亚基的抑制的机制也不清楚。我们进行了分子对接，并预测LDAO结合可能发生在ATP合酶的催化位点，无论该位点是空的还是含有核苷酸。分子动力学模拟表明，LDAO可影响催化位点附近β亚基中环状结构（ATP合酶中的β404 - 415位残基）的流动性。对该酶中β409位残基和ATP合酶中相应的β419位残基进行诱变，结果表明该残基的侧链类型确实影响LDAO对ATP酶活性的刺激作用。我们还发现，与无硫酸盐培养基相比，在100 mM硫酸盐存在下LDAO对该酶的激活作用更强。这种现象可能是由于硫酸盐增强了该酶的ADP抑制作用。
