Purification of the "corrinoid" enzyme involved in the synthesis of acetate by Clostridium thermoaceticum.
A corrinoid enzyme has been purified to approximately 80% homogeneity from Clostridium thermoaceticum. It catalyzes the formation of acetate from N5-methyltetrahydrofolate and pyruvate in combination with the required supplementary enzymes which are supplied by an extract that has been treated with propyl iodide. The enzyme was purified by chromatography on a folate affinity column and a DEAE-Bio-Gel column and by ultrafiltration. The molecular weight as determined by sedimentation equilibrium is 158,000 and the sedimentation coefficient is 10.5 S. By gel electrophoresis in sodium dodecyl sulfate, the subunit molecular weight was found to be 40,000, thus, the enzyme may be a tetramer of four similar subunits. The results of electron microscopy confirmed the tetrameric structure. In the absence of sodium dodecyl sulfate, two bands of similar intensity were observed by electrophoresis, but both yielded the 40,000 molecular weight subunit in the presence of sodium dodecyl sulfate. These results indicate the two bands represent either two different molecular weight forms of the enzyme or two differently charged isoenzymes. The enzyme is quite labile being sensitive to dilution, aerobic conditions, and light. Dithiothreitol and glycerol were found to stabilize the enzyme. The cofactor requirements for acetate synthesis have been determined. ATP, thiamin pyrophosphate, S-adenosylmethionine, and Fe2+ were found to be required for maximum activity and the Km values were determined. High concentrations of methyltetrahydrofolate, pyruvate, and S-adenosylmethionine were found to inhibit the synthesis of acetate.
D000085:Acetates / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D007700:Kinetics; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism; D011773:Pyruvates
D000085:Acetates; D009097:Multienzyme Complexes; D011773:Pyruvates; D008780:Methyltransferases