Arch Microbiol (1992) 157:284-289 Archives of Hicrobiology © Springer-Verlag 1992 A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii Mike S. M. Jetten, Tineke J. Fluit, Alfons J. M. Stams, and Alexander J. B. Zehnder Department of Microbiology, Wageningen Agricultural University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands Received October 10, 1991/Accepted November 6, 1991 Abstract. An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homo- geneity. The molecular mass estimated by gelfiltration was 139 _+ 7 kDa. Sodium dodecyl sulfate/polyacryl- amide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an ~2/~2 oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri- and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg 2+ was required for activity. The pH optimum was 8 at 1 mM PPi and 5 mM Mg 2+. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent Km for PPi of 0.1 mM in the presence of 5 mM Mg 2÷. The Vmax was 590 pmol of pyrophosphate hy- drolyzed per min per mg protein, which corresponds to a Kcat of 1400 per second. The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedure were applied. Key words: Methanothrix soehngenii - Acetate degrada- tion - Energetics - Inorganic pyrophosphatase - Fluoride inhibition Methanothrix soehngenii (M. soehngenii) uses solely ace- tate as carbon and energy source (Zehnder et al. 1980; Huser et al. 1982). The growth yield (1.4 g/tool acetate) and the growth rate (average doubling time of 7 days) are low, but the affinity (Ks = 0.5 mM) for acetate of M. soehngenii is high (Huser et al. 1982; Jetten et al. Offprint requests to: A. J. B. Zehnder Abbrevation: PMSF, phenylmethylsulfonyl fluoride 1990b). In M. soehngenii acetate is activated to acetyl- CoA by acetyl-CoA synthetase [1] (Jetten et al. 1989b). Ac+ATP + HSCoA ~ Ac-SCoA+AMP+PPi [1] AMP + ATP ~ 2 ADP [21 PPi + H20 ~ 2 Pi [3] Ac+2ATP+HSCoA ~ AcSCoA +2ADP + 2P~ [4] The formed AMP is converted to ADP by adenylate kinase [2] and pyrophosphate is hydrolyzed by pyro- phosphatase [3] (Jetten et al. 1989b). The sum of these reactions [4] indicates that acetate in M. soehngenii is activated to acetyl-CoA at the expense of two energy rich phosphate-bonds (Jetten et al. 1989b). Because the con- version of acetyl-CoA to methane is supposed to be coupled to the synthesis of 1 < ATP < 2 (Thauer et al. 1989), it is not likely that M. soehngenii will just hydrolyze pyrophosphate. For a favourable energy balance the energy from the pyrophosphate bond has to be conserved. Therefore, the inorganic pyrophosphatase of M. soehn- genii was investigated. Enzymes relatively specific for the hydrolysis of in- organic pyrophosphate are widely distributed in nature. Inorganic pyrophosphatases have been purified from heterotrophic (Josse 1966a; Wong et al. 1970; Josse and Wong 1971; Hachimori et al. 1975; Lathi and Niemi i98I; Lathi 1983), chemolithoautotrophic (Tominaga and Mori 1977), sulfate reducing (Ware and Postgate 1971) and phototrophic bacteria (Klemme et al. 1971; Randahl 1979) and from yeasts (Kunitz and Robbins 1961). The isolation and characterization of this enzyme from an archae is not yet reported. The purification and some of the properties of inorganic pyrophosphatase from M. soehngenii are described here. Materials and methods Gases, chemicals, and biochemicals All chemicalswereat least of analyticalgrade. Triton X-305, tri- and tetrapolyphosphate were purchased from Sigma (Amsterdam,The