Factors that Influence Recombinant Lysine Deacetylase Activity

Lysine deacetylases (KDACs) are the enzymes that reverse the post translational modification of acetylation, resulting in an unmodified lysine residue and acetate. KDACs play a critical role in the cell and changes to their activity have been linked to diseases. In order to characterize KDAC activity in a biologically relevant fashion, in vitro assay buffer should ideally mimic the in vivo environment. In this project, our research objective is to determine the components of buffer that influence KDAC activity while remaining biologically relevant and cost efficient. The starting buffer used consisted of phosphate, glycerol and potassium chloride. The buffer parameters varied included the viscosity, buffering agent, and metabolite concentrations. The effect of these alterations was measured through the use of fluorescence‐based activity assays. KDAC8 was the primary enzyme used to observe the effects of these variations. Although the variation of most of these parameters either did not affect KDAC8 activity or decreased it, the addition of a metabolite mixture to the reaction slightly increased KDAC8 activity. In order to determine which metabolite caused the activity increase, citrate was selected for further investigation due to its metal chelating properties. KDAC8 and other Class IIa KDACs require a metal cation at their active site to function. In excess, some of these metals can also bind to an inhibitory site on the enzyme. When citrate was added to the reaction in the presence of excess zinc, activity was partially restored. However, the addition of citrate alone had no effect on KDAC8 activity. In conclusion, a minimal phosphate buffer optimizes KDAC8 activity while remaining biologically relevant and cost efficient. Furthermore, the addition of citrate is a biologically relevant method to ensure that excess trace metals are not inhibiting KDAC8. Support or Funding Information U.S. Army Research Laboratory and the U.S. Army Research Office DoD W911NF‐13‐1‐0129, Summer Undergraduate Research Fellowship and the Center for Undergraduate Research at Xavier University of Louisiana.