Expression and Kinetic Characterization of Recombinant Human Stomach Alcohol Dehydrogenase

• Published in: The Journal of biological chemistry 1995-02, Vol.270 (8), p.3625-3630
• Main Authors: Kedishvili, Natalia Y., Bosron, William F., Stone, Carol L., Hurley, Thomas D., Peggs, Cara F., Thomasson, Holly R., Popov, Kirill M., Carr, Lucinda G., Edenberg, Howard J., Li, Ting-Kai
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.8.3625

A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase ( -ADH) was isolated from a human stomach cDNA library. The 373-amino acid -ADH encoded by this cDNA was expressed in Escherichia coli . The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25°C, calculated from active-site titration of NADH binding, was 92 ± 9 units/mg. Kinetic analysis of the catalytic efficiency ( k / K ) of recombinant -ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human -ADH exhibited high catalytic efficiency for oxidation of all- trans -retinol to all- trans -retinal. This pathway is important in the synthesis of the transcriptional regulator all- trans -retinoic acid. Secondary alcohols and 3β-hydroxysteroids were inactive with -ADH or were oxidized with very low efficiency. The K of -ADH for ethanol was 25 mM, and the K for primary straight chain alcohols decreased substantially as chain length increased. There are important amino acid differences in the alcohol-binding site between the human class IV ( ) and human class I (β) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all- trans -retinol, the high k for ethanol, and the low catalytic efficiency for secondary alcohols of -ADH relative to β 1 -ADH. For example, modeling the binding of all- trans -retinol in the human β 1 -ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site. The deletion of Gly-117 in human -ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.