Probing the Role of a Catalytic Residue in a Lysine Deacetylase

Acetylation of lysine is a common post‐translational protein modification. Lysine deacetylases (KDACs) reverse this modification. However, little information is known concerning the role of several conserved active site residues in KDACs. Studies have shown that if the reversal of this modification does not take place correctly, it can lead to many human diseases, including cancers. Recent studies also suggest that an active site residue that is found as either a tyrosine or histidine in human KDACs is important for enzymatic activity; however, how this active site residue functions is not entirely understood. Wild‐type KDAC7 contains a histidine at this position and is generally inactive with known KDAC substrates, whereas wild‐type KDAC8 contains a tyrosine residue and has considerable activity. To begin to understand how this residue affects catalytic activity of the enzyme, we created variants of KDAC7 and KDAC8 containing selected amino acids in this position of the active site, then expressed and purified the variants using both insect and E. coli cells. The resulting proteins were evaluated using several substrates to determine how each mutation affected the activity and substrate specificity of the enzymes. The KDAC variants tested demonstrated distinct specificity profiles for those substrates that have been tested thus far. Circular dichroism spectroscopy was used to analyze the secondary structure of the variants and revealed that reductions of enzyme activity were not due to major structural perturbations. We are currently determining kinetic parameters for the KDAC variants with multiple peptide substrates to determine whether the active site residue affects catalysis, substrate binding, or both. This approach will ultimately further our understanding of the mechanism by which KDACs function to deacetylate their target substrates.