title: General Base-General Acid Catalysis in Human Histone Deacetylase 8.
abstract: Histone deacetylases (HDACs) regulate cellular processes such as differentiation and apoptosis and are targeted by anticancer therapeutics in development and in the clinic. HDAC8 is a metal-dependent class I HDAC and is proposed to use a general acid-base catalytic pair in the mechanism of amide bond hydrolysis. Here, we report site-directed mutagenesis and enzymological measurements to elucidate the catalytic mechanism of HDAC8. Specifically, we focus on the catalytic function of Y306 and the histidine-aspartate dyads H142-D176 and H143-D183. Additionally, we report X-ray crystal structures of four representative HDAC8 mutants: D176N, D176N/Y306F, D176A/Y306F, and H142A/Y306F. These structures provide a useful framework for understanding enzymological measurements. The pH dependence of kcat/KM for wild-type Co(II)-HDAC8 is bell-shaped with two pKa values of 7.4 and 10.0. The upper pKa reflects the ionization of the metal-bound water molecule and shifts to 9.1 in Zn(II)-HDAC8. The H142A mutant has activity 230-fold lower than that of wild-type HDAC8, but the pKa1 value is not altered. Y306F HDAC8 is 150-fold less active than the wild-type enzyme; crystal structures show that Y306 hydrogen bonds with the zinc-bound substrate carbonyl, poised for transition state stabilization. The H143A and H142A/H143A mutants exhibit activity that is >80000-fold lower than that of wild-type HDAC8; the buried D176N and D176A mutants have significant catalytic effects, with more subtle effects caused by D183N and D183A. These enzymological and structural studies strongly suggest that H143 functions as a single general base-general acid catalyst, while H142 remains positively charged and serves as an electrostatic catalyst for transition state stabilization.
authors.affiliation: Departments of Chemistry and Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Sister M Lucy
authors.initials: SM
authors.lastname: Gantt
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Christophe
authors.initials: C
authors.lastname: Decroos
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Lee
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Gullett
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Bowman
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , Philadelphia, Pennsylvania 19104-6323, United States.|Radcliffe Institute for Advanced Study and Department of Chemistry and Chemical Biology, Harvard University , Cambridge, Massachusetts 02138, United States.
authors.forename: David W
authors.initials: DW
authors.lastname: Christianson
authors.affiliation: Departments of Chemistry and Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D000143:Acids / Q000737:chemistry*; D000468:Alkalies / Q000737:chemistry*; D002384:Catalysis; D018360:Crystallography, X-Ray; D006655:Histone Deacetylases / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000143:Acids; D000468:Alkalies; D012097:Repressor Proteins; C409470:HDAC8 protein, human; D006655:Histone Deacetylases