Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme.
Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.
D000596:Amino Acids / Q000032:analysis; D001410:Bacillus megaterium / Q000201:enzymology*; D002850:Chromatography, Gel; D002950:Citrate (si)-Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000378:metabolism*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases