Department of Biology, University of Konstanz, 78464 Konstanz, Germany.
Biochemistry. 2013 Aug 20;52(33):5563-76. doi: 10.1021/bi400729e. Epub 2013 Aug 9.
The high-affinity potassium uptake system KdpFABC is a unique type Ia P-type ATPase, because it separates the sites of ATP hydrolysis and ion transport on two different subunits. KdpFABC was expressed in Escherichia coli. It was then isolated and purified to homogeneity to obtain a detergent-solubilized enzyme complex that allowed the analysis of ion binding properties. The electrogenicity and binding affinities of the ion pump for K(+) and H(+) were determined in detergent-solubilized complexes by means of the electrochromic styryl dye RH421. Half-saturating K(+) concentrations and pK values for H(+) binding could be obtained in both the unphosphorylated and phosphorylated conformations of KdpFABC. The interaction of both ions with KdpFABC was studied in detail, and the presence of independent binding sites was ascertained. It is proposed that KdpFABC reconstituted in vesicles translocates protons at a low efficiency opposite from the well-established import of K(+) into the bacteria. On the basis of our results, various mechanistic pump cycle models were derived from the general Post-Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC.
高亲和力钾摄取系统 KdpFABC 是一种独特的 I 型 P 型 ATP 酶，因为它将 ATP 水解和离子转运的位点分离到两个不同的亚基上。KdpFABC 在大肠杆菌中表达。然后将其分离并纯化至均相，以获得可用于分析离子结合特性的去污剂溶解的酶复合物。用电致变色的 styryl 染料 RH421 在去污剂溶解的复合物中测定离子泵对 K(+)和 H(+)的电生性和结合亲和力。在 KdpFABC 的未磷酸化和磷酸化构象中都可以获得半饱和 K(+)浓度和 pK 值，用于 H(+)结合。详细研究了两种离子与 KdpFABC 的相互作用，并确定了存在独立的结合位点。据推测，在囊泡中重建的 KdpFABC 以低效率将质子逆向转运，与 K(+)进入细菌的既定导入相反。基于我们的结果，从 P 型 ATP 酶的一般 Post-Albers 方案推导出了各种机制泵循环模型，并根据实验证据进行了讨论，以提出 KdpFABC 可能的分子泵循环。
