A Continuous, Nonradioactive Assay for Histone Acetyltransferases

Kim, Youngjoo; Tanner, Kirk; Denu, John M.

doi:10.1006/abio.2000.4546

Cited by 86 publications

(86 citation statements)

References 14 publications

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“…Self-acetylation of purified, minimally acetylated SSAT was carried out at a concentration of 10 M SSAT and 100 M AcCoA in 100 mM NaCl͞10 mM Tris⅐HCl, pH 7.5͞1 mM DTT at 20°C and followed by Western blots by using anti-acetyl-lysine (rabbit antiserum) (Upstate Biotechnology). Acetylation of spermidine or spermine by SSAT was assayed in a coupled reaction originally developed to measure histone acetyltransferase activity (37). The CoA generated in the acetyltransferase reaction is used by pyruvate dehydrogenase to oxidize pyruvate, a reaction that is accompanied by the reduction of NAD ϩ to NADH, which is measured spectrophotometrically at 340 nm.…”

Section: Methodsmentioning

confidence: 99%

Structures of wild-type and mutant human spermidine/spermine N ¹ -acetyltransferase, a potential therapeutic drug target

Bewley

Graziano

Jiang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Spermidine͞spermine N 1 -acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined highresolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N 1 ,N 11 -bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalzyed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine Nacetyltransferase activity.GCN5-related N-acetyltransferase family ͉ polyamines ͉ symmetric and asymmetric dimer ͉ self-acetylation ͉ thialysine N -acetyltransferase

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Section: Methodsmentioning

confidence: 99%

Structures of wild-type and mutant human spermidine/spermine N ¹ -acetyltransferase, a potential therapeutic drug target

Bewley

Graziano

Jiang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…The mutations were verified by DNA sequencing. The catalytic domain (amino acids 99 -262) of yGCN5, and single amino acid substituted forms, were recombinantly expressed in BL21-DE3 bacteria by isopropyl-␤-D-thiogalactopyranoside induction for 4 h at 25°C using the T7 polymerase-based expression system as described (27). Following chromatography on S-Sepharose, fractions containing yGCN5 or the desired mutant protein (assessed by SDS-PAGE and HAT activity) were pooled and concentrated and then subjected to size-exclusion chromatography on G-75 Sephadex.…”

Section: Methodsmentioning

confidence: 99%

Mutational Analysis of Conserved Residues in the GCN5 Family of Histone Acetyltransferases

Langer

Tanner

Denu

2001

Journal of Biological Chemistry

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GCN5 is a critical transcriptional co-activator and is the defining member of a large superfamily of N-acetyltransferases. GCN5 catalyzes the transfer of an acetyl group from acetyl-CoA to the ⑀-amino of lysine 14 within the core H3 histone protein. Previous biochemical analyses have indicated a fully ordered kinetic mechanism. Recent structural studies have implicated several conserved residues in catalysis and substrate binding. Here the roles of Glu-173, His-145, and Asp-214 in yeast GCN5 have been evaluated using site-directed mutagenesis, steady state and pre-steady state kinetics, pH analysis, isotope partitioning, and equilibrium binding studies. The results with wild type and E173Q, H145A, and D214A mutants are consistent with chemical catalysis being rate-determining in turnover. All mutants exhibited K d values (3.5-8.5 M) for AcCoA that were similar to wild type enzyme, indicating no functional role for these residues in AcCoA binding. The E173Q mutant demonstrated a ϳ500 -600-fold decreases in k cat and k cat /K m , H3 , consistent with Glu-173 acting as the general base catalyst as proposed previously. No significant effect was observed on substrate binding steps. His-145 was identified as a residue in the peptide binding cleft that must be unprotonated (pK a ‫؍‬ 5.8) for peptide binding and likely hydrogen-bonds to the Ser-10 hydroxyl of histone H3. His-145 also contributes to lowering the pK a value (by 0.8 units) of general base Glu-173 through a water-mediated hydrogen bond to the carboxylate side chain. Analysis of D214A revealed an obligate protein isomerization step that occurs after AcCoA binding and permits efficient peptide binding. Asp-214 is part of a conformationally flexible loop that mediates the isomerization by stabilizing distinct conformers of the protein.Histone acetyltransferases (HATs) 1 are classified as enzymes capable of transferring an acetyl group from acetyl-CoA to an acceptor histone protein substrate. The acceptor site is the ⑀-amino group of lysine side chains within the aminoterminal tails of the core histones, H2A, H2B, H3, and H4. At least four gene families of HATs have been identified in mammals (1, 2). The largest family includes the defining member GCN5 whose catalytic domain is well conserved from yeast to humans. The GCN5 family of HATs are part of a larger superfamily of enzymes capable of acetyl transfer to amine-containing substrates (referred to as GNATs, for GCN5-like N-acetyltransferases). PCAF (p300/CBP-associating factor) (3-6) is also a member of the GCN5 family of HATs, displaying similar substrate specificity within the catalytic domain. In vitro, GCN5 displays a strong preference for Lys-14 of histone H3, although other acetylation sites on H3 and H4 have been observed (7-10). Yeast GCN5 has been known for some time to be essential for full transcriptional activation in a subset of genes (11-13). After the discovery of HAT activity in a related Tetrahymena enzyme (14), the causal link between histone acetylation and gene activation was codifying ...

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“…In Vitro Steady-state Kinetic Assays-To continuously monitor the acetylation reaction, we utilized a coupled enzymatic assay to measure the kinetic parameters of MaKat, in which CoA, a product of the acetylation reaction, was used by pyruvate dehydrogenase to convert NADϩ to NADH, resulting in an absorbance increase at 340 nm (12,13). This enzyme-coupled assay condition was optimized for each enzyme and substrate to avoid other rate-limiting factors.…”

Section: Site-directed Mutagenesis and Purification Of Micau_0428mentioning

confidence: 99%

“…The acetylation reaction of MaKat was monitored by a coupled enzymatic assay where the amount of CoA that is liberated following acetylation is measured by formation of reduced NADH from NAD ϩ by pyruvate dehydrogenase (12,13). As shown in Fig.…”

Section: Makat Could Function As An Amino Acid-regulated Proteinmentioning

confidence: 99%

Allosteric Regulation of a Protein Acetyltransferase in Micromonospora aurantiaca by the Amino Acids Cysteine and Arginine

You

Leng

et al. 2014

Journal of Biological Chemistry

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Background: Reversible lysine acetylation of proteins is ubiquitous in actinomycetales. Results: Arginine and cysteine allosterically regulate the protein lysine acetyltransferase Micau_1670 in Micromonospora aurantiaca. Conclusion:The amino acid-binding domain is fused to GCN5-related acetyltransferases, conferring amino acid-induced allosteric regulation to these enzymes. Significance: Activities mediated by amino acids in these acetyltransferases directly link amino acid metabolism to cellular acetylation of proteins.

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A Continuous, Nonradioactive Assay for Histone Acetyltransferases

Cited by 86 publications

References 14 publications

Structures of wild-type and mutant human spermidine/spermine N ¹ -acetyltransferase, a potential therapeutic drug target

Structures of wild-type and mutant human spermidine/spermine N ¹ -acetyltransferase, a potential therapeutic drug target

Mutational Analysis of Conserved Residues in the GCN5 Family of Histone Acetyltransferases

Allosteric Regulation of a Protein Acetyltransferase in Micromonospora aurantiaca by the Amino Acids Cysteine and Arginine

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A Continuous, Nonradioactive Assay for Histone Acetyltransferases

Cited by 86 publications

References 14 publications

Structures of wild-type and mutant human spermidine/spermine N 1 -acetyltransferase, a potential therapeutic drug target

Structures of wild-type and mutant human spermidine/spermine N 1 -acetyltransferase, a potential therapeutic drug target

Mutational Analysis of Conserved Residues in the GCN5 Family of Histone Acetyltransferases

Allosteric Regulation of a Protein Acetyltransferase in Micromonospora aurantiaca by the Amino Acids Cysteine and Arginine

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Structures of wild-type and mutant human spermidine/spermine N ¹ -acetyltransferase, a potential therapeutic drug target

Structures of wild-type and mutant human spermidine/spermine N ¹ -acetyltransferase, a potential therapeutic drug target