Fluorophore Conjugation to Substrates Affects Lysine Deacetylase Activity and Specificity

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo . Lysine deacetylases (KDACs) are enzymes that reverse this post‐translational modification, by catalyzing the hydrolysis of ɛ‐N‐acetyllysine residues in proteins via a conserved mechanism. KDAC activity has also been linked to numerous diseases, in particular chronic diseases such as asthma, cancers, muscular disorders, and diabetes. However, details regarding exactly how these enzymes work and KDAC substrate recognition are not well understood. Widely used in vitro systems for studying KDACs involve peptide substrates containing fluorescent dye molecules, such as coumarin. While experimentally convenient for studying enzyme function and testing potential KDAC inhibitors, the impact of these fluorophores on enzyme activity and specificity remain largely unknown. We recently developed a biologically relevant assay that allows us to quantitate deacetylase activity using unlabeled substrates. Using this assay, we determined that the fluorophore enhanced activity with metal‐dependent KDACs and reduced activity with NAD‐dependent KDACs compared to unlabeled substrate sequences. A hydrophobic residue in the C‐terminal position adjacent to the acetyllysine in place of the fluorophore does not mimic the effect of the fluorophore, indicating that the effect is specific to the dye used and is not biologically relevant. Furthermore, the fluorophore does not simply change binding affinity, as we observed changes in both catalytic turnover and substrate specificity. This suggests that the dye molecules affect the KDAC‐substrate interaction in a complex manner, which has significant implications for the interpretation of experiments using fluorophore‐labeled substrates and the biological relevance of results obtained using them.