A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii |
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Authors: | Mike S M Jetten Tineke J Fluit Alfons J M Stams Alexander J B Zehnder |
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Institution: | (1) Department of Microbiology, Wageningen Agricultural University, hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands |
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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
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 Mg2+ was required for activity. The pH optimum was 8 at 1 mM PP
i
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 K
m for PP
i
of 0.1 mM in the presence of 5 mM Mg2+. The V
max was 590 mol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a K
cat 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 procedyre were applied.Abbreviation PMSF
phenylmethylsulfonyl fluoride |
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Keywords: | Methanothrix soehngenii Acetate degradation Energetics Inorganic pyrophosphatase Fluoride inhibition |
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