Purification and characterization of the oxygen-sensitive 4-hydroxybutanoate dehydrogenase from Clostridium kluyveri.
Cell extracts of Clostridium kluyveri grown on ethanol plus succinate contained a NAD(H) dependent 4-hydroxybutanoate dehydrogenase (EC 1.1.1.61) at 66 U/mg. This enzyme was purified 42-fold under anaerobic conditions to homogeneity. Heat treatment, ion exchange chromatography on DEAE-cellulose, nondenaturing polyacrylamide gel electrophoresis, hydrophobic interaction chromatography on phenyl agarose, and gel filtration on Sephadex G-100 were used in the purification. The molecular mass of the enzyme was estimated to be 41.6 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 86 kDa by gel filtration which indicates the active form of the enzyme is dimeric. The protein contains two atoms of Cu and one atom of Fe per monomeric unit. The enzyme exhibits maximum activity at pH 6.1 for the reduction of succinic semialdehyde and at pH 9.4 for the oxidization of 4-hydroxybutanoate. The Km values for NADH and succinic semialdehyde were 150 +/- 20 microM and 560 +/- 80 microM, respectively. In the reverse direction, the Km values were 670 +/- 80 microM and 55 +/- 16 mM for NAD and 4-hydroxybutanoate, respectively. The enzyme is inactivated by oxygen. The inactivation occurs with a t1/2 = 4.5 min at pH 8.2 and 30 degrees C.
Wolff
R A
RA
Department of Biochemistry, University of Wisconsin, Madison 53706, USA.
Kenealy
W R
WR
D000388:Air; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D003013:Clostridium / Q000201:enzymology*; D003300:Copper / Q000302:isolation & purification; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000302:isolation & purification; D007700:Kinetics; D008667:Metalloproteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology*; D011487:Protein Conformation; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; D008667:Metalloproteins; D009243:NAD; D005680:gamma-Aminobutyric Acid; D003300:Copper; D007501:Iron; D006884:Hydroxybutyrate Dehydrogenase; C019416:4-hydroxybutyrate dehydrogenase; C009338:succinic semialdehyde; D010100:Oxygen