5DC7

Crystal structure of D176A-Y306F HDAC8 in complex with a tetrapeptide substrate

Summary for 5DC7

• Entry DOI: 10.2210/pdb5dc7/pdb
• Related: 5DC5 5DC6 5DC8
• Descriptor: Histone deacetylase 8, Fluor-de-Lys tetrapeptide assay substrate, ZINC ION, ... (6 entities in total)
• Functional Keywords: histone deacetylase 8, arginase fold, deacetylase, enzyme-substrate complex, hydrolase
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 4
• Total formula weight: 88595.32

Authors

Decroos, C.,Lee, M.S.,Christianson, D.W. (deposition date: 2015-08-23, release date: 2016-02-03, Last modification date: 2024-10-30)

Primary citation

Gantt, S.M.,Decroos, C.,Lee, M.S.,Gullett, L.E.,Bowman, C.M.,Christianson, D.W.,Fierke, C.A.
General Base-General Acid Catalysis in Human Histone Deacetylase 8.
Biochemistry, 55:820-832, 2016

PubMed 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.

PubMed: 26806311
DOI: 10.1021/acs.biochem.5b01327

Experimental method

X-RAY DIFFRACTION (2.3 Å)