Coupled oxidoreductase activity of horse liver alcohol dehydrogenase

Two assay procedures were used to study ethanol oxidation by crystalline horse liver alcohol dehydrogenase: (A) Acetaldehyde formed by ethanol oxidation (with and without lactaldehyde) was distilled into a semicarbazide solution and the absorbance of the acetaldehyde semicarbazone formed was measured at 224 nm. (B) 1,2-propandiol formed in the coupled oxidation-reduction reaction (CH 2 CH 2 OH + CH 2 · was measured by a colorimetric method. The K M for ethanol oxidation at pH 7.5 was 0.35 m m . With increasing concentrations of lactaldehyde, both the V M and the K M for ethanol oxidation were increased. At different concentrations of lactaldehyde, the ratio K M V M was found to be constant. The K M for lactaldehyde was 1.1 m m and was not affected by addition of ethanol. The K M for NAD + (H) in the coupled O-R reaction was 4.5 μ m . Propanol and butanol could replace ethanol in the coupled O-R reaction, but were inhibitory at high concentrations. Increasing the lactaldehyde concentration partially relieved this inhibition. In addition to lactaldehyde, glyceraldehyde also stimulated ethanol oxidation. The V M and K M values of three alcohol:aldehyde pairs were compared to determine how the substrate nature controls the efficiency of an alcohol:aldehyde pair in the coupled O-R reaction. Several NAD + analogs were tested in the coupled O-R reaction. It was observed that although these analogs were more active than NAD + in ethanol oxidation in the absence of lactaldehyde, they were less active than NAD + when lactaldehyde was present. A mechanism has been proposed to explain the coupled oxidoreductase activity of liver alcohol dehydrogenase. The mechanism postulates that the rate-limiting step in the coupled oxidoreductase reaction is either the ternary complex interconversions (E-NAD + -RCH 2 OH ⇌ E-NADH-RCHO), or the dissociation steps leading to the formation of E-NAD + (H) and product. The mechanism is consistent with the kinetic results described in this paper.