Role of the potassium/lysine cationic center in catalysis and functional asymmetry in membrane-bound pyrophosphatases

Abstract

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.

Roles of the Hydrophobic Gate and Exit Channel in Vigna radiata Pyrophosphatase Ion Translocation

“…M-PPases comprise a 66-90 kDa polypeptide containing 16 transmembrane helices (TMs), and it forms a dimer [2,11,12]. M-PPases are classified into two major types, K + -dependent or K + -independent [13,14], depending on presence of a functional determinant residue (alanine or lysine). K + -independent M-PPases have a conserved lysine that uses the NH3 side-chain group to occupy the K + position for M-PPase activation, and they can be further classified into two subfamilies based on whether or not they are regulated by Na + [15].…”

Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a non-phosphorous allosteric inhibitor

“…These changes increase affinity and activity. They also provide a possible structural explanation for the Na + /H + pumping enzymes 22,34 , where H + -pumping is not inhibited by 100 mM Na + and 50 mM K + . One way this might happen is through a conformation similar to that observed here: the binding of PP i to monomer A and pumping of, say, a proton would drive changes in the loop12-13 and TMH 13 such that the conformation of monomer B became suitable for binding PPi and pumping Na + .…”

“…There is recent evidence of functional asymmetry in K + -dependent mPPases in the presence of excess potassium 22 . Here we show the first structural evidence of asymmetry.…”

“…analysis, we used a variant of Artukka, Luoto22 , which assumed different kinetic behavior of each monomer active sites of the mPPase dimer, with the addition of inhibitor (Scheme 1). E 2 represents the dimeric enzyme, S represents substrate (Na 4 PP i ), I represents inhibitor (ATC), n represent the amount of ATC binds to the enzyme, Ks is a microscopic Michaelis-Menten constant, and k p is per-site maximum rates for the substrate complex.…”

Energy Coupling in Cation-Pumping Pyrophosphatase—Back to Mitchell

“…An alternative possibility is that Na + and H + transport are carried out by different subunits of dimeric Na + -PPase binding Na + at a single site per subunit in a negatively cooperative manner because of dimer asymmetry (Artukka et al, 2018;Vidilaseris et al, 2019) ( Figure 1F). In this mechanism, Na + could inhibit H + transport by occupying both pump-loading sites, resembling the effect of high substrate concentration on enzymatic activity (Artukka et al, 2018).…”