Protein engineering of alcohol dehydrogenase -- 1. Effects of two amino acid changes in the active site of yeast ADH-1

One of the promises held out by protein engineering is the abilityto alter predictably the properties of an enzyme to enable itto find new substrates or catalyse existing substrates moreefficiently, such manipulations being of interest both enzymologicallyand, potentially, industrially. It has been postulated thatin yeast alcohol dehydrogenase (YADH-1) certain amino acidssuch as Trp 93 and Thr 48 constrict the active site due to theirbulky side chains and thus impede catalysis of molecules largerthan ethanol. To study effects of enlarging the active sitewe have made two changes into YADH-1, replacing Trp 93 withPhe and Thr 48 with Ser. Kinetic experiments showed that thisenzyme had marked increases in reaction velocity for the n-alcoholspropanol, butanol, pentanol, hexanol, heptanol, octanol andcinnamyl alcohol compared to the parent, agreeing with the predictionthat expanding the active site should facilitate the oxidationof larger alcohols. The substrate affinities were slightly reducedin the altered enzyme, possibly due to its having reduced hydrophobicityat Phe 93.