<br/>Acetylation of lysine is a common post-translational protein modification. Lysine deacetylases (KDACs) reverse this modification and therefore contribute to protein regulation, and incorrect control of acetylation contributes to many human diseases. Recent studies have demonstrated that an active site residue, found as either a tyrosine or histidine in human KDACs, is important for enzymatic activity. Wild-type KDAC7 contains a histidine at this position and is generally inactive with synthetic KDAC substrates. Wild-type KDAC8 contains a tyrosine residue and has considerable activity. Prior work established that a histidine-to-tyrosine mutation at that position in KDAC7 results in an increase in activity. To begin to understand how and why this residue affects catalytic activity of the enzyme, we created variants of KDAC7 and KDAC8 containing selected mutations in the active site at this position using site-directed mutagenesis, then expressed and purified the variants. The enzymatic activity of the resulting proteins were evaluated using biologically relevant substrates to determine how each mutation affected the activity and substrate specificity of the enzymes. Kinetic parameters indicate that the affinity of active peptides varies at least 20-fold with mutated KDAC7, a substantially larger variation than is observed with KDAC8 with the same peptides. A detectable change in the secondary structure was observed indicating a structural change with this mutation in KDAC7, but not KDAC8, which also may account for the increase in activity. The mutated KDAC7 not only has an increase in activity, but also reacts with a wider variety of peptides. Ongoing work to prepare and characterize variants with additional amino acid mutations at this position will further understanding of the importance of this site in KDACs.

Exploring substrates for lysine deacetylase 7

255th National Meeting (2018)

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