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

Natalia Y. Kedishvili, William F. Bosron, Carol L. Stone, Thomas D. Hurley, Cara F. Peggs, Holly R. Thomasson, Kirill M. Popov, Lucinda G. Carr, Howard J. Edenberg, Ting Kai Li

Research output: Contribution to journalArticle

75 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)3625-3630
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number8
DOIs
StatePublished - Feb 24 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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