Purification and steady-state kinetic characterization of human liver beta 3 beta 3 alcohol dehydrogenase

Biochemistry. 1989 Aug 22;28(17):6810-5. doi: 10.1021/bi00443a005.

Abstract

Human liver alcohol dehydrogenase catalyzes the NAD+-dependent oxidation of alcohols. Isoenzymes are produced in liver by five different genes, two of which are polymorphic. We have studied the three beta beta isoenzymes produced at ADH2 because they exhibit very different kinetic properties and they appear with different frequencies in different racial populations. The beta 3 beta 3 isoenzyme which appears in 25% of black Americans was purified to homogeneity, and conditions were found to stabilize this labile isoenzyme. The comparison of substrate specificity among beta beta isoenzymes for primary straight-chain alcohols indicates that there is a positive correlation between Vmax/KM and the log octanol/water partition coefficient for alcohols with beta 2 beta 2 and beta 3 beta 3 but not with beta 1 beta 1. Methyl substitutions at C1 or C2 of these alcohols reduce the catalytic efficiency with all three isoenzymes. The KM and Ki values of beta 3 beta 3 for NAD+ and NADH are substantially higher than values for beta 1 beta 1 or beta 2 beta 2. The Vmax of beta 3 beta 3 ethanol oxidation is 90 times that of beta 1 beta 1. Sequencing of the beta 3 subunit and gene indicates that the polymorphism results from a single amino acid exchange of Cys-369 in beta 3 for Arg-369 in beta 1 and beta 2 [Burnell et al. (1987) Biochem. Biophys. Res. Commun. 146, 1227-1233]. In horse alcohol dehydrogenase and beta 1 beta 1, the guanidino group of Arg-369 is thought to stabilize the NAD(H)-enzyme complex by bonding to one of the pyrophosphate oxygens.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alcohol Dehydrogenase / isolation & purification*
  • Alcohol Dehydrogenase / metabolism
  • Chromatography, Affinity
  • Chromatography, DEAE-Cellulose
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Electrophoresis, Starch Gel
  • Humans
  • Isoelectric Focusing
  • Isoenzymes / isolation & purification*
  • Isoenzymes / metabolism
  • Kinetics
  • Liver / enzymology*
  • Molecular Weight
  • Polymorphism, Genetic
  • Regression Analysis
  • Substrate Specificity

Substances

  • Isoenzymes
  • Alcohol Dehydrogenase