title: A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii.
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 homogeneity. The molecular mass estimated by gelfiltration was 139 +/- 7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an alpha 2 beta 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 Mg2+ was required for activity. The pH optimum was 8 at 1 mM PPi and 5 mM Mg2+. 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 Mg2+. The Vmax was 590 mumol of pyrophosphate hydrolyzed 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.
authors.affiliation: Department of Microbiology, Wageningen Agricultural University, The Netherlands.
authors.forename: M S
authors.initials: MS
authors.lastname: Jetten
authors.forename: T J
authors.initials: TJ
authors.lastname: Fluit
authors.forename: A J
authors.initials: AJ
authors.lastname: Stams
authors.forename: A J
authors.initials: AJ
authors.lastname: Zehnder
mesh_terms: D001105:Archaea / Q000201:enzymology*; D004795:Enzyme Stability; D005459:Fluorides / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011489:Protein Denaturation; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D046911:Macromolecular Substances; D011755:Pyrophosphatases; D005459:Fluorides