Abstract
The human liver alpha alpha alcohol dehydrogenase exhibits a different substrate specificity and stereospecificity for secondary alcohols than the human beta 1 beta 1, and gamma 1 gamma 1 or horse liver alcohol dehydrogenases. All of the enzymes efficiently oxidize primary alcohols, but alpha alpha oxidizes secondary alcohols far more efficiently than human beta 1 beta 1 and gamma 1 gamma 1 or horse liver alcohol dehydrogenase. Specifically, alpha alpha oxidizes four- and five-carbon secondary alcohols with efficiencies 0.06-2.2 times that of primary homologs and oxidizes these secondary alcohols with efficiencies up to 3 orders of magnitude greater than those of the three other isoenzymes. Whereas the human beta 1 beta 1, gamma 1 gamma 1 and horse isoenzymes show a distinct preference toward (S)-(+)-3-methyl-2-butanol, the alpha alpha isoenzyme prefers (R)-(-)-3-methyl-2-butanol. Computer-simulated graphics demonstrate that the horse subunit accommodates (S)-(+)-3-methyl-2-butanol within the active site much better than the opposite stereoisomer, primarily due to steric hindrance caused by Phe-93. Human alpha may accommodate (R)-(-)-3-methyl-2-butanol better than (S)-(+)-3-methyl-2-butanol because of close contacts between the latter and Thr-48. These observations suggest that substitutions at positions 93 and 48 in the active site of human liver alcohol dehydrogenase isoenzymes may determine their substrate specificity for secondary alcohols.
Highlights
The human liver aa alcohol dehydrogenase exhibits alcohol-binding cleft and that may affect substrate specificity a different substrate specificity and stereospecificity include Ser-48, Leu-57, Phe-110, Leu-116, Leu-141, and Ilefor secondary alcohols than the human &Y1, and ylyl or horse liver alcohol dehydrogenases
Michaelis kinetic constant (K,) andcatalytic efficiency ( VmJKm ) of human liver aa, PIP1, ylyl, and horse liver alcohol dehydrogenase were determined toward increasing lengths of primary alcohols having straight and iso-chains (Tables I and 11)
Whereas the V, of primary alcohols was not generally affected by increasing chain length, a variation in the VmaX toward cyclohexanol and 3-methyl-2-butanol was observed
Summary
From the Departments of Biochemistry and Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46223. Horse liver alcohol dehydrogenase (alcoho1:NAD' oxidoreductase, EC 1.1.1.1)exhibits broad substrate specificity and oxidizes secondary as well as primary alcohols [1, 2]. The structure of the substrate-binding site of horse liver alcohol dehydrogenase has been determined by x-ray crystallography of several complexes of the enzyme with coenzyme and substrate (e.g. NAD' and bromobenzyl alcohol [6]), or inhibitors (e.g. NADH and dimethyl sulfoxide [7]; NAD' and pyrazole [8]).These structural studies indicate that the substrate-binding site islined with hydrophobic residues including Phe-93 This bulky amino acid is believed to play an important role in substrate specificity for both primary [6] and secondary [3] alcohols, presumably by sterically blocking the binding of alcohols. Enzyme (0.5-13 pg/ml) and 2.4 mM NAD+in 0.1 M sodiumphosphate, Substrate SpecifiHcituymoafn
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