Substitutions of isoleucine residues at the adenine binding site activate horse liver alcohol dehydrogenase.

The research explores how changes in certain amino acid residues in horse liver alcohol dehydrogenase can increase the enzyme’s ability to bind to an adenine compound and accelerate its catalyzing action. These findings could potentially enhance our understanding of enzyme function and might have applications in enzymatic therapies.

Objective of the Study

• The researchers aimed to investigate the contribution of specific amino acids, isoleucine residues at positions 224 and 269, to the binding capacity and catalysis of the horse liver alcohol dehydrogenase enzyme.

Method used in the Study

• Isoleucine residues at 224 and 269 were replaced with other amino acids – glycine (I224G) and serine (I269S). This is an approach used to mimic the effects of genetic mutations in the protein.

Key Findings of the Study

• Both mutations resulted in reduced affinities for several adenosine derivatives, with affinity decreasing 5 to 50 times.
• The change at position 269 (I269S) further destabilized the coenzyme binding, reducing affinities for NAD+ and NADH by 60-fold and 350-fold respectively.
• This mutation also enhanced the rate of change in the enzyme’s conformation when NAD+ binds to it.
• Furthermore, it allowed the enzyme to oxidize ethanol at a faster pace (up to 26 times with I269S mutation as compared to up to 7 times with I224G mutation).
• This enhanced oxidation was credited to the faster release of NADH made possible by the I269S mutation.
• However, the limiting factor in the rate of ethanol oxidation was the hydride transfer when I269S was present.
• Despite these changes, the inhibition constants for 2,2,2-trifluoroethanol and N-methylformamide – two substrate analogues – and catalytic efficiencies for ethanol and acetaldehyde remained unchanged. This suggests that the adenosine binding to the coenzyme is not directly related to the subsequent reaction of substrates.

Implications of the Research

• The study shows that altering specific amino acid residues can influence enzyme function dramatically. Such findings could be a foundation for further investigation into enzyme modification, potentially informing targeted therapies in medical and industrial fields where enzymes play a key role.