Purification and properties of 4-hydroxycyclohexanecarboxylate dehydrogenase from Corynebacterium cyclohexanicum.
4-Hydroxycyclohexanecarboxylate dehydrogenase, which requires NAD as a cofactor, was detected in crude soluble extracts of Corynebacterium cyclohexanicum grown on cyclohexanecarboxylic acid as the sole carbon source. The dehydrogenase was purified from extracts to an electrophoretically homogenous state by ammonium sulfate precipitation and chromatography on DEAE-650s, agarose-NAD and hydroxyapatite. The enzyme consisted of two identical subunits and had a native relative molecular mass of 53,600. There were two residues each of cysteine and tryptophan in the enzyme molecule. Oxo acid rather than hydroxy acid was routinely used as substrate for assay of the enzyme. The enzyme is highly specific for 4-oxocyclohexanecarboxylic acid: the carboxyl group is essential and the position of carbonyl group is important; neither the 2-oxo nor the 3-oxo homologue was used as substrate. A methyl substitution on the ring of 4-oxocyclohexanecarboxylate resulted in an almost complete loss of its activity. The reduction product was identified as trans-4-hydroxycyclohexanecarboxylic acid by gas-liquid chromatography and mass spectrometry. It was used as a substrate for the reverse reaction in the presence of NAD but not its cis-isomer. The enzyme was specific for the B-side (pro-S) hydrogen of NADH in the hydrogen transfer from NADH to 4-oxocyclohexanecarboxylate. The Km values for 4-oxocyclohexanecarboxylate and NADH in the reduction reaction at pH 6.8 were 0.50 mM and 0.28 mM, respectively, whereas those for trans-4-hydroxycyclohexanecarboxylate and NAD in the oxidation reaction at pH 8.8 were 0.51 mM and 0.23 mM, respectively. The equilibrium constant of the reaction was 1.79 x 10(-10) M. The enzyme was strongly inhibited by N-bromosuccinimide.
Obata
H
H
Alberta Research Council, Edmonton, Canada.
Uebayasi
M
M
Kaneda
T
T
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D002848:Chromatography, DEAE-Cellulose; D003352:Corynebacterium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D009243:NAD; D000429:Alcohol Oxidoreductases; C021395:trans-4-hydroxycyclohexanecarboxylic acid dehydrogenase