Expression and kinetic characterization of recombinant human stomach alcohol dehydrogenase: Active-site amino acid sequence explains substrate specificity compared with liver isozymes

Academic Article

Abstract

  • A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (σ-ADH) was isolated from a human stomach cDNA library. The 373-amino acid σ-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 (kcat/KM) of recombinant σ-ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human σ-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 σ-ADH or were oxidized with very low efficiency. The KM of σ-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 (σ) and human class I (β) 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 σ-ADH relative to β1-ADH. For example, modeling the binding of all-trans-retinol in the human β1-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 σ-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.
  • Published In

    Digital Object Identifier (doi)

    Author List

  • Kedishvili NY; Bosron WF; Stone CL; Hurley TD; Peggs CF; Thomasson HR; Popov KM; Carr LG; Edenberg HJ; Li TK
  • Start Page

  • 3625
  • End Page

  • 3630
  • Volume

  • 270
  • Issue

  • 8