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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. We report 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 thatmore » 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.« less

Authors:
 [1];  [2];  [3];  [4];  [5];  [4];  [6]
  1. Univ. of Michigan, Ann Arbor, MI (United States); Franciscan Univ. of Steubenville, OH (United States)
  2. Univ. of Pennsylvania, Philadelphia, PA (United States); Aix Marseille Univ., Centrale Marseille (France)
  3. Univ. of Pennsylvania, Philadelphia, PA (United States)
  4. Univ. of Pennsylvania, Philadelphia, PA (United States); Harvard Univ., Cambridge, MA (United States)
  5. Univ. of Pennsylvania, Philadelphia, PA (United States); Univ. of California, San Francisco, CA (United States)
  6. Univ. of Michigan, Ann Arbor, MI (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1238290
Grant/Contract Number:  
GM40602; GM49758
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 55; Journal Issue: 5; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; Peptides and proteins; Monomers; Molecules; Catalysts; Noncovalent interactions

Citation Formats

Gantt, Sister M. Lucy, Decroos, Christophe, Lee, Matthew S., Gullett, Laura E., Bowman, Christine M., Christianson, David W., and Fierke, Carol A. General Base–General Acid Catalysis in Human Histone Deacetylase 8. United States: N. p., 2016. Web. doi:10.1021/acs.biochem.5b01327.
Gantt, Sister M. Lucy, Decroos, Christophe, Lee, Matthew S., Gullett, Laura E., Bowman, Christine M., Christianson, David W., & Fierke, Carol A. General Base–General Acid Catalysis in Human Histone Deacetylase 8. United States. https://doi.org/10.1021/acs.biochem.5b01327
Gantt, Sister M. Lucy, Decroos, Christophe, Lee, Matthew S., Gullett, Laura E., Bowman, Christine M., Christianson, David W., and Fierke, Carol A. 2016. "General Base–General Acid Catalysis in Human Histone Deacetylase 8". United States. https://doi.org/10.1021/acs.biochem.5b01327. https://www.osti.gov/servlets/purl/1238290.
@article{osti_1238290,
title = {General Base–General Acid Catalysis in Human Histone Deacetylase 8},
author = {Gantt, Sister M. Lucy and Decroos, Christophe and Lee, Matthew S. and Gullett, Laura E. and Bowman, Christine M. and Christianson, David W. and Fierke, Carol A.},
abstractNote = {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. We report 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.},
doi = {10.1021/acs.biochem.5b01327},
url = {https://www.osti.gov/biblio/1238290}, journal = {Biochemistry},
issn = {0006-2960},
number = 5,
volume = 55,
place = {United States},
year = {Mon Jan 25 00:00:00 EST 2016},
month = {Mon Jan 25 00:00:00 EST 2016}
}

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Works referencing / citing this record:

Structure–activity relationships of hydroxamate-based histone deacetylase-8 inhibitors: reality behind anticancer drug discovery
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HDAC3 and HDAC8 are required for cilia assembly and elongation
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Histone deacetylase 6 structure and molecular basis of catalysis and inhibition
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Formation of Unstable and very Reactive Chemical Species Catalyzed by Metalloenzymes: A Mechanistic Overview
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Factors governing when a metal-bound water is deprotonated in proteins
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Histone deacetylase 6 structure and molecular basis of catalysis and inhibition
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HDAC3 and HDAC8 are required for cilia assembly and elongation
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Formation of Unstable and very Reactive Chemical Species Catalyzed by Metalloenzymes: A Mechanistic Overview
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