Acetyl-coenzyme A synthetase in the thermophilic, acetate-utilizing methanogen Methanothrix sp. strain CALS-1 |
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Authors: | Yok Lan Teh Stephen Zinder |
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Institution: | Section of Microbiology, Wing Hall, Cornell University, Ithaca, New York, USA |
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Abstract: | Abstract There is considerable evidence that acetyl-CoA synthetase (acetate thiokinase, ACS, EC 6.2.1) is responsible for acetate activation in the mesophilic acetotrophic methanogen Methanothrix soehngenii . If the pyrophosphate produced by ACS is simply cleaved, two high-energy phosphodiester bonds are expended in acetate activation. Hi High ACS activity (2–4 μmol min−1 mg protein−1) was present in cell-free extracts of the thermophile Methanothrix sp. strain CALS-1. The 23-fold purified enzyme had a molecular mass near 165 kDa and a subunit molecular mass near 78 kDa, suggesting that the enzyme is a homodimer. The temperature optimum for ACS was near 70°C and the apparent K m values were 2–4 mM for acetate and 5.5 mM for MgATP. Coenzyme A at concentrations greater than 0.2 mM inhibited ACS, while acetyl-CoA was not inhibitory. AMP and pyrophosphate inhibited ACS with K i values of 5 mM and 1.5 mM respectively. Other divalent cations could replace Mg2+, with Mn2+ showing the highest activity. Activity with ITP was 20% of that with ATP, and other nucleotides tested were considerably less active. Since Methanothrix sp. strain CALS-1 has an active soluble pyrophosphatase, it appears that it uses the same energetically costly method for acetate activation as M. soehngenii . |
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Keywords: | Methanothrix sp strain CALS-1 Acetyl-CoA synthetase Methanogenesis |
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