Substrate Specificity of Metal-Dependent Lysine Deacetylases.

Abstract

Lysine deacetylases (KDACs) catalyze the deacetylation of acetylated lysine residues on histones and other protein substrates. This modification plays a vital role in numerous cellular processes and inhibitors targeting KDAC are effective in a variety of diseases. Eleven members of the 18-member KDAC family require a divalent metal ion for activity. Previous studies have demonstrated that the reactivity of KDAC8 varies with the bound metal ion (Co2+> Fe2+ > Zn2+). Here I show that the bound metal ion also regulates the substrate specificity of KDAC8. Incubation of metal-substituted KDAC8 with an acetylated peptide library identified peptides that were deacetylated more rapidly by either Zn2+- or Fe2+-KDAC8 and peptide substrates that display similar reactivity with both enzymes. The steady-state kinetic parameters measured for peptides from each specificity profile showed that the kcat/KM values recapitulate the results observed in the library screen confirming the metal-dependent substrate selectivity. These studies suggest that KDAC8 activity and specificity are regulated in vivo by Zn/Fe metal switching.

Furthermore, the reactivity of KDAC11, with peptide libraries demonstrates that the activity and specificity of this isozyme are also dependent on the bound metal ion. Furthermore, these selectivity screens led to the identification of a peptide motif (GPK(ac)LGC) that corresponds to a sequence near the N-terminus of Cdt1 (IIAPPKLACRTPS), a proposed in vivo substrate for KDAC11. The value of the kcat/KM for the Zn2+-bound KDAC11 on this sequence is increased by 1333-fold over the commercially available coumarin substrate to a value of 0.33 ± 0.04 µM-1s-1 and the reactivity with Fe2+-KDAC11 is even higher (>0.5 µM-1S-1).

Finally I show that Arg37, a residue proposed to regulate the dissociation of acetate in KDAC8, is critical for the catalytic activity of KDAC8. Substitution of Arg37 with either alanine (R37A) or glutamate (R37E) decreases the value of kcat/KM by 528-fold and 4 x 105-fold, respectively. The circular dichroism spectrum show that the secondary structure of KDAC8 is not affected by the R37A mutation but is altered by the R37E mutation. These results show that Arg37 is important for catalysis and acetate affinity, and could be exploited for preparing specific inhibitors of KDAC8.