title: Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii.
abstract: In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.
authors.affiliation: Department of Microbiology, Agricultural University Wageningen, The Netherlands.
authors.forename: M S
authors.initials: MS
authors.lastname: Jetten
authors.forename: A J
authors.initials: AJ
authors.lastname: Stams
authors.forename: A J
authors.initials: AJ
authors.lastname: Zehnder
mesh_terms: D000106:Acetate-CoA Ligase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000085:Acetates / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000302:isolation & purification*; D011756:Diphosphates / Q000494:pharmacology; D019605:Euryarchaeota / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D000085:Acetates; D011756:Diphosphates; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase