Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Biológica and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Buenos Aires, Argentina.
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Biológica and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Buenos Aires, Argentina.
Biochim Biophys Acta Biomembr. 2019 Feb 1;1861(2):355-365. doi: 10.1016/j.bbamem.2018.10.020. Epub 2018 Nov 7.
Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeF) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeF is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, k, for the formation of E2P decreases with [Pi] whereas that of E2BeF increases with [BeF]. This might wrongly be taken as evidence of a mechanism where the binding of BeF induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeF follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na. Although E2P and E2BeF are able to form states with 2 occluded Rb, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb is much lower in E2BeF than in E2P. The higher rates of Rb occlusion and deocclusion observed for E2BeF, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeF mimics the E2P ground state.
金属氟化物配合物已被用于诱导 E2P 类似态，目的是研究 P 型 ATP 酶中 E2P 水解过程中发生的事件。在本工作中，我们比较了铍氟化物配合物（BeF）诱导的 E2P 类似态与 Na,K-ATP 酶后门磷酸化形成的实际 E2P 态。通过使用 styryl 染料 RH421 研究了 E2P 和 E2P 类似态的形成。我们发现，只有氟化膦酸盐类似物 BeF 像 Pi 一样，能增加 RH421 的荧光。E2P 形成的观察到的速率常数 k 随 [Pi]的降低而降低，而 E2BeF 的形成速率常数 k 随 [BeF]的增加而增加。这可能会被错误地视为 BeF 结合诱导构象转变机制的证据。在这里，我们提出，与 Pi 一样，BeF 的结合遵循构象选择机制，即它结合到 E2 构象形成一个比 E2P 更稳定的复合物，从其在加入 Na 后返回 E1 的能力受损就可以看出。尽管 E2P 和 E2BeF 都能够形成具有 2 个结合的 Rb 的状态，但这两种酶复合物的不同之处在于，E2BeF 对第二个 Rb 的结合和结合的亲和力比 E2P 低得多。与其他 E2P 类似的过渡态和产物态相比，E2BeF 观察到的 Rb 结合和去结合的更高速率表明，阳离子转运位点的开放程度更高，这支持了 E2BeF 模拟 E2P 基态的观点。
