title: Characterization of the two catalytic domains in histone deacetylase 6.
abstract: Histone deacetylase 6 (HDAC6) is the only known HDAC with two potentially functional catalytic domains, yet the role towards substrate played by these two domains remains ambiguous. Most studies report HDAC6 activities measured using either immune complexes or in vitro translated products. Here, we characterize the activity of highly purified recombinant HDAC6, mutants with active site histidine mutations in each domain (H216A and H611A), and individual catalytic domains. The deacetylase activities of these proteins, as well as their kinetic parameters, were measured using histone, alpha-tubulin, and fluorogenic acetylated lysine as substrates. Mutant H216A only slightly lowers the catalytic rate. However, mutant H611A decreases the catalytic rate more than 5000-fold. The first domain expressed alone is not catalytically active. In contrast, the second domain shows only a modest decrease in substrate binding and product formation rate. Our results indicate that the in vitro deacetylase activity of HDAC6 resides in the C-terminal second catalytic domain.
authors.affiliation: Takeda San Diego Inc., CA 92121, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Zou
authors.forename: Yiqin
authors.initials: Y
authors.lastname: Wu
authors.forename: Marc
authors.initials: M
authors.lastname: Navre
authors.forename: Bi-Ching
authors.initials: BC
authors.lastname: Sang
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D006657:Histones / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014404:Tubulin / Q000737:chemistry*
chemical_list: D006657:Histones; D014404:Tubulin; C433811:HDAC6 protein, human; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases; D008239:Lysine