Kinetics and Comparative Reactivity of Human Class I and Class IIb Histone Deacetylases
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- 作者：Brian E. Schultz ; Shawn Misialek ; Jiansheng Wu ; Jie Tang ; Marion T. Conn ; Ram Tahilramani ; and Lance Wong
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：August 31, 2004
- 年：2004
- 卷：43
- 期：34
- 页码：11083 - 11091
- 全文大小：115K
- 年卷期：v.43,no.34(August 31, 2004)
- ISSN：1520-4995
文摘
Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation ofacetylated lysine residues on histone proteins. They operate in biological systems as part of multiproteincorepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactorbinding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10were measured using a novel, continuous protease-coupled enzyme assay. Values of kcat and kcat/Km andthe pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N
-acetyl-Lys)-AMC. Values of kcat spanned the range of 0.006-2.8 s-1, and kcat/Km values rangedfrom 60 to 110000 M-1 s-1. The pH profiles for both kcat and kcat/Km were bell-shaped for all of theHDAC isozymes, with pH optima at approximately pH 8. Values of Ki for the inhibitor trichostatin Awere determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes,except that the value for HDAC8 was significantly higher than that for the other isozymes. The reactionof HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussedin terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysinehydrolysis by these enzymes.
-acetyl-Lys)-AMC. Values of kcat spanned the range of 0.006-2.8 s-1, and kcat/Km values rangedfrom 60 to 110000 M-1 s-1. The pH profiles for both kcat and kcat/Km were bell-shaped for all of theHDAC isozymes, with pH optima at approximately pH 8. Values of Ki for the inhibitor trichostatin Awere determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes,except that the value for HDAC8 was significantly higher than that for the other isozymes. The reactionof HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussedin terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysinehydrolysis by these enzymes.