Acetate Activation inMethanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase

Abstract

The thermophilic methanogenMethanosaeta thermophilauses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction inMethanosarcina sp.is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction inMt. thermophilaseems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate(PPi)and was only moderately inhibited byPPi. The breakdown ofPPiwas performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part ofPPi(KM=0.27±0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides orPPi. However, it cannot be excluded that otherPPi-dependent enzymes take advantage of the remainingPPiand contribute to the energy balance of the cell.