Active Site Metal Identity Alters Histone Deacetylase 8 Substrate Selectivity: A Potential Novel Regulatory Mechanism

Abstract

Histone deacetylase 8 (HDAC8) is a well-characterized member of the class I acetyl-lysine deacetylase (HDAC) family. Previous work has shown that the efficiency of HDAC8-catalyzed deacetylation of a methylcoumarin peptide varies depending on the identity of the divalent metal ion in the HDAC8 active site. Here we demonstrate that both HDAC8 activity and substrate selectivity for a diverse range of peptide substrates depend on the identity of the active site metal ion. Varied deacetylase activities of Fe(II)- and Zn(II)-HDAC8 toward an array of peptide substrates were identified using self-assembled monolayers for matrix-assisted laser desorption ionization (SAMDI) mass spectrometry. Subsequently, the metal dependence of deacetylation of peptides of biological interest was measured using an in vitro peptide assay. While Fe(II)-HDAC8 is generally more active than Zn(II)-HDAC8, the Fe(II)/Zn(II) HDAC8 activity ratio varies widely (from 2 to 150) among the peptides tested. These data provide support for the hypothesis that HDAC8 may undergo metal switching in vivo that, in turn, may regulate its activity. However, future studies are needed to explore the identity of the metal ion bound to HDAC8 in cells under varied conditions.

Figure 1. Heat maps visualizing HDAC8 activity and selectivity

The selectivity of (A) Zn(II)- and (B) Fe(II)-bound HDAC8 was determined by applying each metal-substituted enzyme form to an array of 361 peptides of the sequence GXK^AcZGC. The extent of deacetylation of each peptide is shown in grey scale on the heatmaps. (C) A metal-dependent peptide selectivity heat map was generated by taking the ratio of Zn(II)-HDAC8 to Fe(II)-HDAC8 product conversion for each peptide. Peptides with a greater than seven-fold preference for Zn(II)-HDAC8 are shown in red and Fe(II)-selective peptides are shown in blue. Peptides that were deacetylated similarly by both enzyme forms are shown in grey and peptides that were not deacetylated (<3%) by either enzyme form are shown in white.

Figure 2. Representative peptide assay data

The dependence of the initial rate on substrate concentration for Fe(II)-(■) and Zn(II)- (●) HDAC8-catalyzed deacetylation of SMC3 10-mer peptide measured using the acetate assay. A hyperbola was fit to the data to calculate the Michaelis-Menten parameters

Figure 3. Fe(II)/Zn(II) reactivity ratios of various peptide substrates with HDAC8

Metal reactivity and selectivity varied significantly with the sequence of the peptide. Commercially available methylcoumarin bound substrates were used as controls. HDAC8 p53 FdL data are reported in ().