Purification and properties of acetyl-CoA synthetase from Bradyrhizobium japonicum bacteroids.
Acetyl-CoA synthetase was purified 800-fold from Bradyrhizobium japonicum bacteroids. A specific activity of 16 mumol/min per mg of protein was achieved, with a 30-40% yield. The purification scheme consisted of only three consecutive chromatography steps. The enzyme has a native Mr of 150,000, estimated by gel-permeation chromatography, and a subunit Mr of 72,000, determined by SDS/polyacrylamide-gel electrophoresis. The optimum pH and temperature are 8.5 and 50 degrees C respectively. The Km values for acetate, CoA and ATP were 146, 202 and 275 microM respectively. The reaction was specific for acetate, as propionate and oleate were used very poorly. Likewise, the enzyme used only ATP, ADP or dATP; AMP, GTP, XTP and UTP could not replace ATP. Acetyl-CoA synthetase showed a broad specificity for metals; MnCl2 could replace MgCl2. In addition, CaCl2 and CoCl2 were approx. 50% as effective as MgCl2, but FeCl3, NiCl2 or ZnCl2 could not effectively substitute for MgCl2. The enzyme may be regulated by NADP+ and pyruvate; no effect was seen of amino acids, glucose catabolites, reduced nicotinamide nucleotides or acetyl-CoA. Inhibition was seen with AMP, PPi, FMN and pyridoxal phosphate, with Ki values of 720, 222, 397 and 1050 microM respectively.
Preston
G G
GG
Department of Biochemistry and Interdisciplinary Plant Biotechnology, University of Missouri-Columbia 65211.
Wall
J D
JD
Emerich
D W
DW
D000106:Acetate-CoA Ligase / Q000302:isolation & purification* / Q000378:metabolism; D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000255:Adenosine Triphosphate / Q000378:metabolism; D002413:Cations, Divalent; D002845:Chromatography; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D012230:Rhizobiaceae / Q000201:enzymology*; D013379:Substrate Specificity
D000085:Acetates; D002413:Cations, Divalent; D009711:Nucleotides; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase; D019342:Acetic Acid; D003065:Coenzyme A