Chelatable trace zinc causes low, irreproducible KDAC8 activity

Abstract

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.

Lysine Deacetylase Substrate Selectivity: Distinct Interaction Surfaces Drive Positive and Negative Selection for Residues Following Acetyllysine

“…We were primarily interested in KDAC8 (class I) and KDAC6 (class II). Both are relatively well-characterized KDACs with respect to crystal structures and previous screening work, and both have several putative substrates identified from in vitro and/or cell-based experiments. ,,,,− ,− We used KDAC1 for comparison as it is also a moderately well-characterized class I KDAC; however, it is primarily thought to reside in the nucleus and deacetylate histone proteins. ,, We used the FRK ac RW peptide as a starting point. This peptide was derived from an acetylated human protein (ADAP1; UniProtKB O75689), and we previously identified it as an in vitro substrate for KDAC1, KDAC6, and KDAC8. , Using a fluorescamine-based in vitro assay, we tested the activity of the KDACs with a set of peptides derived from FRK ac RW, where we replaced each of the residues following the acetyllysine (the +1 and +2 positions) with alanine and/or swapped their order to determine whether these residues influenced the activities of KDAC1, KDAC6, and KDAC8 with the peptide substrates.…”

Critical review of non‐histone human substrates of metal‐dependent lysine deacetylases

“…Ideally, a direct enzyme‐substrate relationship would be determined by presenting purified enzyme with a purified, recombinant substrate; however, the logistical challenges involved in obtaining an adequate quantity of recombinant substrate protein that is site‐specifically acetylated render this approach not broadly feasible. Additionally, experimental conditions have been shown to have significant effects on KDAC activity in vitro; 14,102,103 thus, it is difficult to determine how well the in vitro conditions mimic the cellular environment and how the behavior of the enzyme in vitro relates to its cellular behavior. Therefore, experiments of both types (cell‐based and in vitro) identifying the same enzyme‐substrate pair are necessary to show direct, biologically relevant deacetylation of a target protein.…”