title: Expression and kinetic characterization of recombinant human stomach alcohol dehydrogenase. Active-site amino acid sequence explains substrate specificity compared with liver isozymes.
abstract: A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (sigma-ADH) was isolated from a human stomach cDNA library. The 373-amino acid sigma-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 degrees C, calculated from active-site titration of NADH binding, was 92 +/- 9 units/mg. Kinetic analysis of the catalytic efficiency (kcat/KM) of recombinant sigma-ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human sigma-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 3 beta-hydroxysteroids were inactive with sigma-ADH or were oxidized with very low efficiency. The KM of sigma-ADH for ethanol was 25 mM, and the KM 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 (sigma) and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high kcat for ethanol, and the low catalytic efficiency for secondary alcohols of sigma-ADH relative to beta 1-ADH. For example, modeling the binding of all-trans-retinol in the human beta 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 sigma-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.
authors.affiliation: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
authors.forename: N Y
authors.initials: NY
authors.lastname: Kedishvili
authors.forename: W F
authors.initials: WF
authors.lastname: Bosron
authors.forename: C L
authors.initials: CL
authors.lastname: Stone
authors.forename: T D
authors.initials: TD
authors.lastname: Hurley
authors.forename: C F
authors.initials: CF
authors.lastname: Peggs
authors.forename: H R
authors.initials: HR
authors.lastname: Thomasson
authors.forename: K M
authors.initials: KM
authors.lastname: Popov
authors.forename: L G
authors.initials: LG
authors.lastname: Carr
authors.forename: H J
authors.initials: HJ
authors.lastname: Edenberg
authors.forename: T K
authors.initials: TK
authors.lastname: Li
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013270:Stomach / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase