Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates

Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Deacetylation of ε-acetyl-lysine residues within the N-terminal tail of core histones mediates changes in both histone–DNA and histone–non-histone protein interactions. Howev...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2004-11, Vol.324 (3), p.1116-1123
Main Authors: Riester, Daniel, Wegener, Dennis, Hildmann, Christian, Schwienhorst, Andreas
Format: Article
Language:English
Subjects:
Acetylpolyamines
Animals
Binding Sites
Bordetella - metabolism
Bordetella/Alcaligenes FB188
Catalytic Domain
Chromatography, High Pressure Liquid
Cloning, Molecular
DNA - chemistry
Fluorogenic assay
Histone deacetylase
Histone Deacetylases - chemistry
Histone Deacetylases - metabolism
Histones - chemistry
Humans
Immunoblotting
Kinetics
Lysine - chemistry
Models, Chemical
Oligonucleotide Probes - chemistry
Polyamines - chemistry
Propionates - pharmacology
Protein Binding
Protein Structure, Tertiary
Pseudomonas aeruginosa
Pseudomonas aeruginosa - metabolism
Rats
Recombinant Proteins - chemistry
Repressor Proteins - metabolism
Sodium Acetate - pharmacology
Sodium Chloride - chemistry
Sodium Chloride - pharmacology
Substrate Specificity
Time Factors
Trifluoroacetic Acid - metabolism
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Summary:Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Deacetylation of ε-acetyl-lysine residues within the N-terminal tail of core histones mediates changes in both histone–DNA and histone–non-histone protein interactions. However, surprisingly little is known about the substrate specificities of different HDACs. Here, we use the ε-acyl moieties of ε-modified l-lysine in peptidic substrates as a probe to examine the active site cavity of HDACs and HDAC-like enzymes. Measurements were based on a fluorogenic assay with small synthetic substrates. Four different enzyme preparations were used derived from rat, human, and bacterial sources. None of the enzymes was able to utilize substrates with ε-acyl moieties larger than acetyl, except rat liver HDAC, which was the only enzyme to convert a substrate containing ε-propionyl- l-lysine. All enzymes exhibited a distinct enantioselectivity toward l-lysine-containing substrates except FB188 HDAH which also deacetylated Boc- d-Lys(ε-acetyl)-MCA. Moreover, all enzymes also exhibited a distinct specificity for the length of the lysine side chain; acetylated ornithine, which comprises one CH 2 unit less in the side chain, was not a substrate. In line with these results, only acetylcadaverin the metabolic degradation product of lysine but neither acetylputrescine (degradation product of ornithine) nor acetylspermidine strongly inhibited enzyme activity. Boc- l-Lys(ε-trifluoroacetyl)-MCA was observed to be a superior substrate for FB188 HDAH, Pseudomonas aeruginosa HDAH (PA3774), and particularly HDAC 8 compared to rat liver HDAC, and is the first suitable (synthetic) substrate for (human-derived) HDAC 8 reported to date. Altogether, the results reveal clear differences in substrate specificity between different HDACs as analyzed in the fluorogenic HDAC assay. Finally, we present the first candidates for HDAC-type-selective substrates that may be useful as biochemical tools to establish the function of particular pathways and to elucidate the role of distinct HDAC subtypes in cellular differentiation and cancer.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2004.09.155