EXPRESSION AND KINETIC CHARACTERIZATION OF RECOMBINANT HUMAN STOMACH ALCOHOL-DEHYDROGENASE - ACTIVE-SITE AMINO-ACID-SEQUENCE EXPLAINS SUBSTRATE-SPECIFICITY COMPARED WITH LIVER ISOZYMES

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 (k(cat)/K-M) 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 K-M of a-ADH for ethanol was 25 mM, and the K-M 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 k(cat) 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.