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 dehyd rogenase (sigma-ADH) was isolated from a human stomach cDNA library. T he 373-amino acid sigma-ADH encoded by this cDNA was expressed in Esch erichia coli, The specific activity of the recombinant enzyme for etha nol 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 oxi dation of primary alcohols indicated broad substrate specificity, Reco mbinant human sigma-ADH exhibited high catalytic efficiency for oxidat ion of all-trans-retinol to all-trans-retinal. This pathway is importa nt in the synthesis of the transcriptional regulator all-trans-retinoi c acid, Secondary alcohols and 3 beta-hydroxysteroids were inactive wi th sigma-ADH or were oxidized with very low efficiency, The K-M of a-A DH for ethanol was 25 mM, and the K-M for primary straight chain alcoh ols decreased substantially as chain length increased. There are impor tant amino acid differences in the alcohol-binding site between the hu man class IV (sigma) and human class I (beta) alcohol dehydrogenases t hat appear to explain the high catalytic efficiency for all-trans-reti nol, 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 st ructure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entranc e 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 facilita te retinol access to the active-site zinc.