Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis.

This study focuses on the differences between the E and S isoenzymes of horse liver alcohol dehydrogenase, particularly on the fact that only the S isoenzyme is active with 3 beta-hydroxysteroids. The abstract highlights the creation of chimeric enzymes that could help to connect the evolution of catalytic activity, showing that several residues indirectly contribute to catalysis.

Key Points

• The E and S isoenzymes of horse liver alcohol dehydrogenase are only different in 10 amino acid residues, however, only the S isoenzyme shows catalytic activity on 3 beta-hydroxysteroids.
• The functional difference between the E and S enzymes is traced back to their structural differences. This was done by creating a series of chimeric enzymes that potentially represent evolution stages of catalytic activity.
• An important discovery was the creation of E/D115 delta enzyme acted upon by removing Asp-115 from the E isoenzyme. This change activated the enzyme on steroids by modifying the substrate binding pocket and moving Leu-116.
• A chimeric enzyme designated “ESE,” containing four changes in or near the substrate binding pocket, was found to be significantly more catalytically efficient on uncharged steroids than the altered E/D115 delta enzyme.
• Molecular modeling suggests the substitutions at residues 94 and 110 have indirect effects on steroid activity. In fact, the ESE enzyme was found to be more active on neutral steroids, not due to alterations in the substrate binding pocket but to other substitutions.
• The abstract also reports on the K366E and Q17E/A43T/A59T substitutions in the S isoenzyme, which led to noticeable improvements in catalytic activity on steroids, but decreases in binding strength and efficiency with small alcohols.
• Tests with specific substances showed that these enzymes were notably more efficient with 1-hexanol and 5 beta-androstane-3 beta,17 beta-diol, than with ethanol.
• The overall conclusion of the research is that several residues not directly participating in substrate binding or chemical catalysis indeed contribute noticeably to catalytic efficiency.