Abstract
Molecular genetic studies have indicated that alcohol dehydrogenase may be involved in the synthesis of retinoic acid, a hormonal molecule regulating diverse cellular functions at the transcriptional level. Class IV alcohol dehydrogenase (ADH) has been reported to be the most efficient enzyme catalyzing oxidation of retinol in human ADH family. Initial velocity, product inhibition, and dead-end inhibition experiments were performed with the recombinant human class IV ADH to elucidate kinetic mechanism with all-trans-retinol and all-trans-retinal as natural substrates. Fluorescence quenching was titrated in formation of the binary and abortive ternary enzyme complexes. The minimal mechanism deduced from steady-state kinetic and equilibrium binding studies is best described as an asymmetric rapid equilibrium random mechanism with two dead-end ternary complexes for retinol oxidation and a rapid equilibrium ordered mechanism with one dead-end ternary complex for retinal reduction, a unique mechanistic form for zinc-containing ADHs in the medium chain dehydrogenase/reductase superfamily. Dissociation constants for the binary complexes as well as the productive and abortive ternary complexes determined from different experimental approaches are in reasonable agreement. Kinetic isotope effect studies suggest rate-limiting isomerization of the central ternary complexes in both reaction directions. The potential interference of retinol metabolism by ethanol through the ADH pathway may play a significant role in the pathogenesis of fetal alcohol syndrome and alcohol-related upper digestive tract cancer.
Highlights
Molecular genetic studies have indicated that alcohol dehydrogenase may be involved in the synthesis of retinoic acid, a hormonal molecule regulating diverse cellular functions at the transcriptional level
NADH versus retinol product inhibition pattern fit linear noncompetitive inhibition (Fig. 1C). These results indicate that the oxidation of retinol is consistent with either a rapid equilibrium random mechanism with abortive ternary enzyme-NADϩ-retinal and enzyme-NADH-retinol complexes or a Theorell-Chance ordered mechanism
These results suggest that the reduction of retinal conforms to either a rapid equilibrium ordered mechanism with an abortive enzyme-NADH-retinol complex or a TheorellChance ordered mechanism
Summary
Anol competitively inhibits retinol oxidation catalyzed by the human ADH family with apparent Ki values ranging from 37 M to 11 mM, indicating that retinoic acid formation in the embryo can be effectively impeded by physiologically attainable levels of blood alcohol during moderate to heavy drinking (16, 32). Human ADH4 is unique in having a deletion of residue 117, which shortens the loop comprised of residues 114 –120 and widens the outer part of the cylinder-shaped substrate-binding pocket as revealed in x-ray studies (38). To evaluate the functional role of ADH in the metabolism of retinoids as well as to assess the potential influence by ethanol consumption in the cell, it is important to understand complete steady-state kinetic mechanism with all-trans-retinol and alltrans-retinal. Both are major metabolites of -carotene and precursors of all-trans-retinoic acid, a ligand for retinoic acid receptors in the retinoid signaling pathway (41)
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