Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russia.
Biochem J. 2018 Mar 26;475(6):1141-1158. doi: 10.1042/BCJ20180071.
Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H and/or Na ions, are divided into K,Na-independent, Na-regulated, and K-dependent families. The first two families include H-transporting mPPases (H-PPases), whereas the last family comprises one Na-transporting, two Na- and H-transporting subfamilies (Na-PPases and Na,H-PPases, respectively), and three H-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K-independent mPPases. Here, we performed a systematic analysis of the K/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K-dependent mPPases abolished the K dependence of hydrolysis and transport activities and decreased these activities close to the level (4-7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K,Na-independent mPPases conferred partial K dependence on the enzyme by unmasking an otherwise conserved K-binding site. Na could partially replace K as an activator of K-dependent mPPases and the Lys → Ala variants of K,Na-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.
膜结合焦磷酸酶（mPPases）通过焦磷酸盐水解将 H 和/或 Na 离子跨膜转运偶联在一起，分为 K、Na 非依赖性、Na 调节性和 K 依赖性家族。前两个家族包括 H 转运 mPPases（H-PPases），而最后一个家族包括一个 Na 转运、两个 Na 和 H 转运亚家族（分别为 Na-PPases 和 Na，H-PPases），以及三个 H 转运亚家族。早期对少数可用模型 mPPases 的研究表明，K 结合到位于焦磷酸盐结合位点附近的一个位点，但在 K 非依赖性 mPPases 中，被进化获得的赖氨酸残基的 ε-氨基取代。在这里，我们对所有 mPPase 亚家族的 K/Lys 阳离子中心进行了系统分析。K 依赖性 mPPases 中的 Ala→Lys 替换消除了水解和转运活性对 K 的依赖性，并使这些活性降低到野生型酶在不存在单价阳离子时观察到的水平（4-7%）。相比之下，K、Na 非依赖性 mPPases 中的 Lys→Ala 替换使酶对 K 具有部分依赖性，从而揭示了一个原本保守的 K 结合位点。Na 可以部分取代 K 作为 K 依赖性 mPPases 的激活剂，以及 K、Na 非依赖性 mPPases 的 Lys→Ala 变体。最后，我们发现所有 mPPases都被过量底物抑制，这表明这些同源二聚体酶的活性位点功能具有强烈的负协同性；此外，K/Lys 中心被确定为该效应机制的一部分。这些发现表明，mPPase 同源二聚体具有活性位点性能的不对称性，这可能是 F 型 ATP 酶旋转结合变化机制的古老原型。
