Purification and characterization of human liver beta 1 beta 1, beta 2 beta 2 and beta Ind beta Ind alcohol dehydrogenase isoenzymes.
Mammalian alcohol dehydrogenases (ADH) are dimeric molecules and electrophoretically distinct isoenzymes appear in human liver. They arise from the combination of different subunits produced at five separate gene loci and polymorphic subunits are produced at two of the loci. Three different homodimeric alloenzymes, beta 1 beta 1, beta 2 beta 2 and beta Ind beta Ind, that are assigned to the ADH2 gene have been purified to homogeneity, and the study of their kinetic properties has revealed striking differences among them. Whereas the pH-optimum for ethanol oxidation of beta 1 beta 1 is 10.0-10.5, that of beta 2 beta 2 is 8.5-8.8 and that of beta Ind beta Ind is 7.0-7.2. The Km of beta 1 beta 1 for ethanol at pH 7.5 is low, 49 microM, in contrast to values for beta 2 beta +2 and beta Ind beta Ind, 0.94 and 64 mM. Km and Ki values for NAD are 7.4 and 90 microM for beta 1 beta 1, 180 and 340 microM for beta 2 beta 2 and 490 and 1300 microM for beta Ind beta Ind. The activity of beta 1 beta 1 is stimulated by 40 mM NaCl but that of beta 2 beta 2 is not. Vmax values for ethanol oxidation range from 9.2 min-1 for beta 1 beta 1 to 400 and 560 min-1 for beta 2 beta 2 and beta Ind beta Ind. Both beta 1 beta 1 and beta 2 beta 2 are inactivated by iodoacetate with the formation of one S-carboxymethylcysteine per subunit; however, the second-order rate constant for inactivation of beta 1 beta 1 is four times that of beta 2 beta 2. These differences in the properties of beta 1 beta 1 and beta 2 beta 2 are consistent with the recently reported substitution of His in beta 2 for Arg 47 in beta 1 at the anion binding center of the active site. The active site sequence of the beta Ind subunit has not yet been determined.
D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D001665:Binding Sites; D004592:Electrophoresis, Starch Gel; D006801:Humans; D066298:In Vitro Techniques; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*
D007461:Iodoacetates; D007527:Isoenzymes; D000429:Alcohol Oxidoreductases; D007460:Iodoacetamide