Alcohol dehydrogenase has been purified from human liver by affinity chromatography. Ultracentrifugation, Sephadex G-200 chromatography, and amino acid analyses of multiple preparations demonstrate homogeneity of molecular weight. Sodium dodecyl sulfate disc gel electrophoresis reveals a single species of molecular weight 42 000. Based on a molecular weight of 85 000 for the dimer obtained from the amino acid composition and a molar absorptivity of A280nm0.1% = 0.58, the enzyme contains 3.6-4.2 g-atoms of zinc, as determined by emission spectrography, microwave-induced emission, and atomic absorption spectrometry. Inhibition by o-phenanthroline, (ethylenedinitrilo)tetraacetic acid, and alpha,alpha'-bipyridine demonstrates that zinc is essential to enzymatic function. Detailed kinetic analyses using primary alcohols of the homologous series CH3(CH2)nOH, n = 0-5, and the corresponding aldehydes as substrates show that KM values become smaller as n increases. This suggest that hydrophobic interactions play a role in substrate binding. The availability of well-defined preparations of human liver alcohol dehydrogenase now allows definitive genetic and functional studies of this enzyme to elucidate human ethanol metabolism.
Interacting selectively and non-covalently with a nucleotide, any compound consisting of a nucleoside that is esterified with (ortho)phosphate or an oligophosphate at any hydroxyl group on the ribose or deoxyribose.
Alcohol dehydrogenase has been purified from human liver by affinity chromatography. Ultracentrifugation, Sephadex G-200 chromatography, and amino acid analyses of multiple preparations demonstrate homogeneity of molecular weight. Sodium dodecyl sulfate disc gel electrophoresis reveals a single species of molecular weight 42 000. Based on a molecular weight of 85 000 for the dimer obtained from the amino acid composition and a molar absorptivity of A280nm0.1% = 0.58, the enzyme contains 3.6-4.2 g-atoms of zinc, as determined by emission spectrography, microwave-induced emission, and atomic absorption spectrometry. Inhibition by o-phenanthroline, (ethylenedinitrilo)tetraacetic acid, and alpha,alpha'-bipyridine demonstrates that zinc is essential to enzymatic function. Detailed kinetic analyses using primary alcohols of the homologous series CH3(CH2)nOH, n = 0-5, and the corresponding aldehydes as substrates show that KM values become smaller as n increases. This suggest that hydrophobic interactions play a role in substrate binding. The availability of well-defined preparations of human liver alcohol dehydrogenase now allows definitive genetic and functional studies of this enzyme to elucidate human ethanol metabolism.
J. Biol. Chem. 265, 16366-16372 (1990)[PubMed:2398055]
Arg-47 of human beta 1 beta 1 alcohol dehydrogenase has been replaced with Lys, His, Gln, and Gly by site-directed mutagenesis. The mutated enzymes were expressed in Escherichia coli and purified to homogeneity. The recombinant enzymes with Arg and His at position 47 exhibit kinetic constants and stability which are similar to beta 1 beta 1 and beta 2 beta 2, respectively. The substitution of Lys, His, or Gln for Arg-47 resulted in active enzymes with lower affinity for coenzyme and higher Vmax values than beta 1 beta 1. The substitution of Gln at position 47 resulted in an enzyme with the highest Vmax for ethanol oxidation of any mammalian alcohol dehydrogenase. In this series of enzymes, the affinity for coenzyme decreases with decreasing pKa of the substituted amino acid side chains. The substitution of Gly at position 47 resulted in an enzyme with a Vmax that was one-half that of the low activity beta 1 beta 1 and coenzyme affinities that are lower than beta 1 beta 1, but are equal to or greater than the affinities exhibited by the His-47 or Gln-47 enzymes. Product inhibition studies indicated a change in mechanism from ordered Bi Bi for beta 1 beta 1 to rapid equilibrium random Bi Bi for the Gly-47 enzyme. The kinetic properties of the Gly-47 enzyme are substantially different from human liver alpha alpha which also has Gly at position 47.
There are 7 different ADH's isozymes in human: three belongs to class-I: alpha, beta, and gamma, one to class-II: pi, one to class-III: chi, one to class-IV: ADH7 and one to class-V: ADH6.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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