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 anti-cancer 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 230-fold lower activity than 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 over 80,000-fold lower than wild-type HDAC8; the buried D176N and D176A mutants have significant catalytic effects, with more subtle effects from 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.

Structural and Functional Influence of the Glycine-Rich Loop G302GGGY on the Catalytic Tyrosine of Histone Deacetylase 8

“…This represents a 500-fold loss of catalytic efficiency, which is comparable to the 150-fold loss of catalytic efficiency observed for Y306F HDAC8 in which the phenolic hydroxyl group is deleted altogether from the aromatic side chain at position 306. 13 …”

“…Both of these interactions are required to polarize the carbonyl for nucleophilic attack by a metal-bound solvent molecule, and both interactions are important for transition state stabilization. 9,12,13 Additionally, a tandem histidine pair is required for general acid-general base catalysis and transition state stabilization. 12,13 In HDAC8, H143 serves both general base and general acid functions, with H142 serving as an electrostatic catalyst (Figure 1).…”

Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8

“…18 Among the metal-dependent HDACs, HDAC8 was the first to yield a crystal structure. 19,20 Important catalytic residues in the HDAC8 active site include tandem histidines (electrostatic catalyst H142 and general base-general acid H143) 21,22 and a conformationally-flexible 23 tyrosine residue that assists the Zn 2+ ion in the polarization of the substrate carbonyl group. 24,25 …”

“…22 This residue functions in catalysis with a positively charged imidazolium group that serves as an electrostatic catalyst. 22 Electron density is observed for the Nε-H proton of H142 in the 1.24 Å electron density map (Supporting Information Figure S7), so the H142 imidazolium group must similarly stabilize the zinc-bound gem-diolate.…”

Metal-dependent Deacetylases: Cancer and Epigenetic Regulators

“…19 The conserved Asp–Asp–His triad coordinates a divalent metal ion that coordinates the metal–water nucleophile. His143 acts as both a general acid and a general base, while Tyr306 and His142 stabilize the oxyanion intermediate.…”