Abstract
The thermophilic autotrophMethanobacterium thermoautotrophicum assimilates CO2 via a novel pathway rather than via the Calvin cycle. The central intermediate of this pathway is acetyl CoA which is reductively carboxylated to pyruvate. Cell extracts of the organism contained phosphoenolpyruvate synthetase with a specific activity of 100 nmol min-1 mg-1 protein (65°C). Pyruvate kinase and pyruvate, phosphate dikinase were not detected. Phosphoenolpyruvate synthetase was partially purified (50-fold) and the following reaction stoichiometry was established:
The enzyme activity was depedent on free Mg2+ ions, NH +4 or K+ ions, and SH-groups. Mn2+, but not Ca2+, could partially substitute for Mg2+; Na+ could not substitute for K+ or NH +4 . The pH-optima,V max-values and the apparentK M-values for the substrates of the enzyme in both directions were determined. Thermodynamic, kinetic and regulatory features indicate that, in vivo, the enzyme functions in the direction of phosphoenolpyruvate synthesis from pyruvate. Not only is the synthesis of phosphoenolpyruvate via the PEP synthetase reaction energetically favorable; the enzyme also catalyzed this synthesis 100 times faster than the reverse reaction, the apparentK M value for pyruvate (40 μM) being low and the apparentK M value for phosphate (100 mM) being high. Furthermore, AMP, ADP, PP and α-ketoglutarate were inhibitors of PEP synthesis, indicating that the enzyme activity may be controlled in vivo. The role of phosphoenolpyruvate synthetase in autotrophic CO2 assimilation pathway ofMethanobacterium, as expected from previous labelling studies, is confirmed.
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Abbreviations
- PEP:
-
Phosphoenolpyruvate
- DTE:
-
dithioerythritol
- DTT:
-
dithiothreitol
- TES:
-
N-tris-(hydrymethyl)methyl-2-aminoethanesulfonic acid
- tricine:
-
N-tris-(hydroxymethyl)methylglycine
- HFPES:
-
N-2-hydroxyethylpiperazine-N-ethanesulfonic acid
- PP:
-
pyrophosphate
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Eyzaguirre, J., Jansen, K. & Fuchs, G. Phosphoenolpyruvate synthetase inMethanobacterium thermoautotrophicum . Arch. Microbiol. 132, 67–74 (1982). https://doi.org/10.1007/BF00690820
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DOI: https://doi.org/10.1007/BF00690820