General Base–General Acid Catalysis in Human Histone Deacetylase 8

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• 作者：Sister M. Lucy Gantt FSGM ; Christophe Decroos ; Matthew S. Lee ; Laura E. Gullett ; Christine M. Bowman ; David W. Christianson ; Carol A. Fierke
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：February 9, 2016
• 年：2016
• 卷：55
• 期：5
• 页码：820-832
• 全文大小：594K
• ISSN：1520-4995

文摘

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 k<sub>catsub>/K<sub>Msub> for wild-type Co(II)-HDAC8 is bell-shaped with two pK<sub>asub> values of 7.4 and 10.0. The upper pK<sub>asub> 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 pK<sub>a1sub> 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.