Toro Tasha B, Edenfield Samantha A, Hylton Brandon J, Watt Terry J
Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA.
Anal Biochem. 2018 Jan 1;540-541:9-14. doi: 10.1016/j.ab.2017.10.024. Epub 2017 Oct 31.
Acetylation is an important regulatory mechanism in cells, and emphasis is being placed on identifying substrates and small molecule modulators of this post-translational modification. However, the reported in vitro activity of the lysine deacetylase KDAC8 is inconsistent across experimental setups, even with the same substrate, complicating progress in the field. We detected trace levels of zinc, a known inhibitor of KDAC8 when present in excess, even in high-quality buffer reagents, at concentrations that are sufficient to significantly inhibit the enzyme under common reaction conditions. We hypothesized that trace zinc in solution could account for the observed variability in KDAC8 activity. We demonstrate that addition of chelators, including BSA, EDTA, and citrate, and/or the use of a phosphate-based buffer instead of the more common tris-based buffer, eliminates the inhibition from low levels of zinc as well as the dependence of specific activity on enzyme concentration. This results in high KDAC8 activity that is consistent across buffer systems, even using low concentrations of enzyme. We report conditions that are suitable for several assays to increase both enzyme activity and reproducibility. Our results have significant implications for approaches used to identify substrates and small molecule modulators of KDAC8 and interpretation of existing data.
乙酰化是细胞中的一种重要调控机制，目前人们正致力于识别这种翻译后修饰的底物和小分子调节剂。然而，即使使用相同的底物，赖氨酸脱乙酰酶KDAC8在不同实验设置下所报道的体外活性也不一致，这使得该领域的研究进展变得复杂。我们检测到痕量水平的锌，即使在高质量的缓冲试剂中，当锌过量存在时它是已知的KDAC8抑制剂，其浓度足以在常见反应条件下显著抑制该酶。我们推测溶液中的痕量锌可能是观察到的KDAC8活性变异性的原因。我们证明，添加螯合剂（包括牛血清白蛋白、乙二胺四乙酸和柠檬酸盐）和/或使用基于磷酸盐的缓冲液而非更常用的基于三羟甲基氨基甲烷的缓冲液，可消除低水平锌的抑制作用以及比活性对酶浓度的依赖性。这导致即使使用低浓度的酶，KDAC8在不同缓冲系统中的活性也很高且一致。我们报告了适用于多种测定以提高酶活性和重现性的条件。我们的结果对用于识别KDAC8底物和小分子调节剂的方法以及现有数据的解释具有重要意义。
