Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum.
The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specific activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 microM coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60 degrees C. Kinetic constants were measured in the forward reaction. The apparent Km for pyruvate and coenzyme A were 322 microM and 3.7 microM, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.
Meinecke
B
B
Institut für Mikrobiologie, Universität Göttingen, Federal Republic of Germany.
Bertram
J
J
Gottschalk
G
G
D002852:Chromatography, Ion Exchange / Q000379:methods; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007658:Ketone Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D050743:Pyruvate Synthase; D013379:Substrate Specificity
D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase