several independent enzyme lineages. Site-directed mutagenesis studies facilitated the identification of a specific Glu residue that appears to be central in the transport mechanism. This residue is located in the cytoplasm-membrane interface of transmembrane helix 6 in Na ؉ -PPases but shifted to within the membrane or helix 5 in H ؉ -PPases. These results contribute to the prediction of the transport specificity and K ؉ dependence for a particular membrane PPase sequence based on its position in the phylogenetic tree, identity of residues in the K ؉ dependence signature, and position of the membrane-located Glu residue.

One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophosphate to support cation gradients in membranes. Transport reactions are catalyzed by membrane-integral pyrophosphatases (PPases), which are classified into two homologous subfamilies: H + -transporting (found in prokaryotes, protists, and plants) and Na + -transporting (found in prokaryotes). Transport activities have been believed to require specific machinery for each ion, in accordance with the prevailing paradigm in membrane transport. However, experiments using a fluorescent pH probe and 22 Na + measurements in the current study revealed that five bacterial PPases expressed in Escherichia coli have the ability to simultaneously translocate H + and Na + into inverted membrane vesicles under physiological conditions. Consistent with data from phylogenetic analyses, our results support the existence of a third, dual-specificity bacterial Na + ,H + -PPase subfamily, which apparently evolved from Na + -PPases. Interestingly, genes for Na + ,H + -PPase have been found in the major microbes colonizing the human gastrointestinal tract. The Na + ,H + -PPases require Na + for hydrolytic and transport activities and are further activated by K + . Based on ionophore effects, we conclude that the Na + and H + transport reactions are electrogenic and do not result from secondary antiport effects. Sequence comparisons further disclosed four Na + ,H + -PPase signature residues located outside the ion conductance channel identified earlier in PPases using X-ray crystallography. Our results collectively support the emerging paradigm that both Na + and H + can be transported via the same mechanism, with switching between Na + and H + specificities requiring only subtle changes in the transporter structure.

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.

SUMMARY In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate hydrolysis to active H + transport across biological membranes (H + -pyrophosphatases) have long been known in prokaryotes, plants, and protists. Recent studies have identified two evolutionarily related and widespread prokaryotic relics that can pump Na + (Na + -pyrophosphatase) or both Na + and H + (Na + ,H + -pyrophosphatase). Both these transporters require Na + for pyrophosphate hydrolysis and are further activated by K + . The determination of the three-dimensional structures of H + - and Na + -pyrophosphatases has been another recent breakthrough in the studies of these cation pumps. Structural and functional studies have highlighted the major determinants of the cation specificities of membrane pyrophosphatases and their potential use in constructing transgenic stress-resistant organisms.

Background: Prokaryotic membrane Na ϩ -PPases couple PP i hydrolysis with active Na ϩ transport.