Purification and characterization of a coenzyme-A-dependent succinate-semialdehyde dehydrogenase from Clostridium kluyveri.
Cell extracts of Clostridium kluyveri, grown on ethanol plus succinate contained a succinyl-CoA:CoA transferase (0.28 U/mg), a coenzyme-A-dependent succinate-semialdehyde dehydrogenase (0.73 U/mg) and a NAD(+)-dependent 4-hydroxybutyrate dehydrogenase (0.25 U/mg). The semialdehyde dehydrogenase, which catalyzed the NADPH-dependent reduction of succinyl-CoA to succinate semialdehyde, was purified 59-fold to homogeneity. A molecular mass of 115000 Da was determined for the native enzyme; SDS/PAGE revealed one protein band at 55,000, indicating that the active form is a dimer. The enzyme was highly specific for succinyl-CoA and succinate semialdehyde. The pH optimum was 7.0 for the reduction of succinyl-CoA, and 8.5 for the reverse reaction. Km values were determined for both the forward and reverse directions. The kinetic data suggest a ping-pong mechanism.
Söhling
B
B
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Federal Republic of Germany.
Gottschalk
G
G
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology*; D006884:Hydroxybutyrate Dehydrogenase / Q000302:isolation & purification; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D050644:Succinate-Semialdehyde Dehydrogenase; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D000214:Acyl Coenzyme A; D013386:Succinates; D009249:NADP; D019802:Succinic Acid; C012046:succinyl-coenzyme A; D006884:Hydroxybutyrate Dehydrogenase; C019416:4-hydroxybutyrate dehydrogenase; D000445:Aldehyde Oxidoreductases; D050644:Succinate-Semialdehyde Dehydrogenase