Arg-47 of human β1β1 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 β1β1 and β2β2, 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 β1β1. 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 β1β1 and coenzyme affinities that are lower than β1β1, 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 β1β1 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 αα which also has Gly at position 47.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - Oct 22 1990|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology