Mechanistic analysis of the pump cycle of the KdpFABC P-type ATPase.

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 electro chromic 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. I bacteria, potassium ions are primarily used to maintain turgor pressure. Additionally, cytoplasmic K is essentially involved in pH homeostasis as well as in the activation of several enzymes. Because of the vital requirement to accumulate potassium ions in the cytoplasm, Escherichia coli comprises a set of different specialized potassium transport systems. Under K limiting conditions (i.e., [K]out < 100 μM), the potassium transport systems TrkG/H, Kup, and KtrAB are not able to provide a flow of K into the bacteria that is sufficient to maintain the cytoplasmic concentrations needed for homeostasis. A high affinity potassium uptake system, the KdpFABC complex, is expressed in the case of such a deprivation. In this ion pump, coupling of ATP hydrolysis to ion transport leads to a high affinity uptake of potassium (KM ≤ 2 μM) but only at moderate transport rates [vmax = 150 μmol of Pi (g of protein) −1 min−1] at 37 °C. KdpFABC belongs to the P type ATPase superfamily, but it has a unique subunit composition (Figure 1). P Type ATPases contain in general a central catalytic subunit that facilitates both ion transport and ATP hydrolysis according to the so called Post− Albers pump cycle (cf. Figure 12A). In contrast, the KdpFABC complex consists of four subunits, and the sites of ATP hydrolysis and ion transport are well separated on two different subunits. Only the KdpB subunit exhibits an explicit homology to other P type ATPases and represents the catalytic subunit performing ATP hydrolysis, whereas the KdpA subunit binds and transports K and shows similarities to KcsA like K channel proteins. The KdpB subunit (72 kDa) is comprised of the typical four functional and structural domains of P type ATPases: the transmembrane (TM) domain, the nucleotide binding (N) domain, the phosphorylation (P) domain, and the actuator (A) domain. As in the case of all P type ATPases, the conserved TGE motif was found in the A domain, which is supposed to Figure 1. Schematic drawing of the KdpFABC complex according to Greie and Altendorf.The KdpB subunit, a P type ATPase, has to be coupled to the KdpA subunit, which resembles a bacterial K channel to perform the observed uphill transport of K. N, P, and A denote functional domains of KdpB. For further explanation, see the text.