title: Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity.
abstract: Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.
authors.affiliation: Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland.
authors.forename: Georgiy A
authors.initials: GA
authors.lastname: Belogurov
authors.forename: Anssi M
authors.initials: AM
authors.lastname: Malinen
authors.forename: Maria V
authors.initials: MV
authors.lastname: Turkina
authors.forename: Ulla
authors.initials: U
authors.lastname: Jalonen
authors.forename: Kalle
authors.initials: K
authors.lastname: Rytkönen
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
authors.forename: Reijo
authors.initials: R
authors.lastname: Lahti
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D002414:Cations, Monovalent / Q000737:chemistry; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012964:Sodium / Q000737:chemistry*; D020124:Thermotoga maritima / Q000201:enzymology* / Q000378:metabolism
chemical_list: D002414:Cations, Monovalent; D001224:Aspartic Acid; D001216:Asparagine; D012964:Sodium; D043564:Inorganic Pyrophosphatase; D000409:Alanine; D011188:Potassium