Kinetic Mechanism of the Histone Acetyltransferase GCN5 from Yeast*

The transcriptional coactivator GCN5 from yeast (yGCN5) is a histone acetyltransferase that is essential for activation of target genes. GCN5 is a member of a large family of histone acetyltransferases that are conserved between yeast and humans. To understand the molecular mechanisms of histone/protein acetylation, a detailed kinetic analysis was performed. Bi-substrate kinetic analysis using acetyl-coenzyme A (AcCoA) and an H3 histone synthetic peptide indicated that both substrates must bind to form a ternary complex before catalysis. Product inhibition studies revealed that the product CoA was a competitive inhibitor versus AcCoA. Desulfo-CoA, a dead-end inhibitor, also demonstrated simple competitive inhibition versus AcCoA. Acetylated (Lys14Ac) H3 peptide displayed noncompetitive inhibition against both H3 peptide and AcCoA. These results support a sequential ternary complex (ordered Bi-Bi) kinetic mechanism, where AcCoA binds first, followed by H3 histone. Acetylated (Lys14Ac) H3 product is released first, and CoA is the last product to leave. Also, two methods were developed to measure the binding affinities of AcCoA/CoA for GCN5. Employing the fluorescent CoA analog etheno-CoA (εCoA, 1-N 6-etheno-CoA), aK d for εCoA of 5.1 ± 1.1 μmwas determined by fluorescence anisotropy. This value was similar to the K d value of 8.5 ± 2.6 μmfor AcCoA obtained using equilibrium dialysis and to theK i (inhibition constant) of 6.7 μmfor CoA obtained from steady-state kinetic assays. Together, these data suggest that the acetyl moiety of AcCoA contributes little to the binding energy.

• Publication year

  2000

• Venue

  Journal of Biological Chemistry

• Publication date

  2000-07-21

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/jbc.M002893200PMID 10811654
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Determining the chemical mechanisms of enzyme-catalyzed reactions by kinetic studies.

  2006cited by this paper
• Melatonin biosynthesis: the structure of serotonin N-acetyltransferase at 2.5 A resolution suggests a catalytic mechanism.

  2000cited by this paper
• A continuous, nonradioactive assay for histone acetyltransferases.

  2000cited by this paper
• Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A

  1999cited by this paper
• Expanded Lysine Acetylation Specificity of Gcn5 in Native Complexes*

  1999cited by this paper
• Crystal structure of the histone acetyltransferase domain of the human PCAF transcriptional regulator bound to coenzyme A

  1999cited by this paper
• Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide

  1999cited by this paper
• The ADA Complex Is a Distinct Histone Acetyltransferase Complex in Saccharomyces cerevisiae

  1999cited by this paper
• Catalytic Mechanism and Function of Invariant Glutamic Acid 173 from the Histone Acetyltransferase GCN5 Transcriptional Coactivator*

  1999cited by this paper
• Structure and function of the core histone N-termini: more than meets the eye.

  1998cited by this paper
• Structure of the histone acetyltransferase Hat1: a paradigm for the GCN5-related N-acetyltransferase superfamily.

  1998cited by this paper
• Kinetic Analysis of the Catalytic Mechanism of Serotonin N-Acetyltransferase (EC 2.3.1.87)*

  1998cited by this paper
• Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase

  1998cited by this paper
• Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.

  1997cited by this paper
• Acetylation of general transcription factors by histone acetyltransferases.

  1997cited by this paper
• Kinetic studies on the binding of 1,N6-etheno-NAD+ to glutamate dehydrogenase from Clostridium symbiosum.

  1997cited by this paper
• Histone acetylation in chromatin structure and transcription

  1997cited by this paper
• Histone acetylation: chromatin in action.

  1997cited by this paper
• Steroid receptor coactivator-1 is a histone acetyltransferase

  1997cited by this paper
• Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation.

  1996cited by this paper
• A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A

  1996cited by this paper
• The CBP co-activator is a histone acetyltransferase

  1996cited by this paper
• A fluorometric assay for measurement of mono-ADP-ribosyltransferase activity.

  1996cited by this paper
• Fluorescence polarization analysis of protein-DNA and protein-protein interactions.

  1996cited by this paper
• Analysis of the Structural Properties of cAMP-responsive Element-binding Protein (CREB) and Phosphorylated CREB*

  1996cited by this paper
• Replacement of catalytic histidine-195 of chloramphenicol acetyltransferase: evidence for a general base role for glutamate.

  1994cited by this paper
• Time‐resolved fluorescence and computational studies of adenylylated glutamine synthetase: Analysis of intersubunit interactions

  1993cited by this paper
• Two distinct yeast transcriptional activators require the function of the GCN5 protein to promote normal levels of transcription.

  1992cited by this paper
• Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes.

  1989cited by this paper
• Fluorescence stopped-flow studies on the binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase.

  1988cited by this paper
• Interactive binding between the substrate and allosteric sites of carbamoyl-phosphate synthetase.

  1988cited by this paper
• A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

  1976cited by this paper
• Enzyme kinetics

  1975cited by this paper

• Activation of Pyruvate Dehydrogenase Complex with Inhibition of Krebs Cycle and Mitochondrial Respiration by the Excess of Pyruvate

  2026cites this paper
• Perioperative neurocognitive disorders: Advances in molecular mechanisms and bioactive molecules

  2025cites this paper
• Growth phase diets diminish histone acetyltransferase Gcn5 function and shorten lifespan of Drosophila males

  2025cites this paper
• Dynamic global acetylation remodeling during the yeast heat shock response

  2025cites this paper
• Redefining the Role of the EryM Acetyltransferase in Natural Product Biosynthetic Pathways

  2025cites this paper
• Redefining the role of the EryM acetyltransferase in natural product biosynthetic pathways.

  2025cites this paper
• Simple and universal function of acetic acid to overcome the drought crisis

  2023cites this paper
• Emerging role of GCN5 in human diseases and its therapeutic potential.

  2023cites this paper
• The multiple facets of acetyl-CoA metabolism: Energetics, biosynthesis, regulation, acylation and inborn errors.

  2022cites this paper
• Modulation of cellular processes by histone and non-histone protein acetylation

  2022cites this paper
• Norepinephrine, beyond the Synapse: Coordinating Epigenetic Codes for Memory

  2022cites this paper
• Metabolic Regulation of Lysine Acetylation: Implications in Cancer.

  2022cites this paper
• Protein acetylation and deacetylation in plant-pathogen interactions.

  2021cites this paper
• Epigenetic Mechanisms in Memory and Cognitive Decline Associated with Aging and Alzheimer’s Disease

  2021cites this paper
• Current Awareness

  2021cites this paper
• Moonlighting Metabolic Enzymes in Cancer: New Perspectives on the Redox Code

  2020cites this paper
• Harnessing Ionic Selectivity In Acetyltransferase Chemoproteomic Probes

  2020cites this paper
• Catalysis by protein acetyltransferase Gcn5.

  2020cites this paper
• The application of histone deacetylases inhibitors in glioblastoma

  2020cites this paper
• Mechanistic and structural studies into the biosynthesis of the bacterial sugar pseudaminic acid (Pse5Ac7Ac).

  2020cites this paper
• The Ada2/Ada3/Gcn5/Sgf29 histone acetyltransferase module.

  2020cites this paper
• Partial Replacement of Nucleosomal DNA with Human FACT Induces Dynamic Exposure and Acetylation of Histone H3 N-Terminal Tails

  2020cites this paper
• Lysine Acetyltransferase Inhibitors From Natural Sources

  2020cites this paper
• The evolving metabolic landscape of chromatin biology and epigenetics

  2020cites this paper
• GCN5 ACETYLTRANSFERASE IN CELLULAR ENERGETIC AND METABOLIC PROCESSES.

  2020cites this paper
• Histone Acetyltransferases: Targets and Inhibitors

  2019cites this paper
• The archaeal Methanocaldococcus infernus Elp3 with N-terminus deletion (1-46)

  2019cites this paper
• Epigenetic regulation by endogenous metabolite pharmacology.

  2019cites this paper
• Mechanisms, Detection, and Relevance of Protein Acetylation in Prokaryotes

  2019cites this paper
• Bm-iAANAT3: Expression and characterization of a novel arylalkylamine N-acyltransferase from Bombyx mori.

  2019influential citation
• Enzymatic and nonenzymatic protein acetylations control glycolysis process in liver diseases

  2019cites this paper
• The Elongator subunit Elp3 is a non-canonical tRNA acetyltransferase

  2019cites this paper
• Investigating the role of electrostatic interactions in modulating protein function

  2018cites this paper
• Metabolismo del acetato en Escherichia coli : caracterización y regulación

  2018influential citation
• Lysine acetyltransferase inhibitors: structure-activity relationships and potential therapeutic implications.

  2018cites this paper
• Investigation of structure and function of Esco1 and Esco2 acetyltransferases.

  2018cites this paper
• Effects of flanking DNA and a transcriptional activator on the histone acetyltransferase activity of the SAGA complex

  2018influential citation
• Distinct requirements of linker DNA and transcriptional activators in promoting SAGA-mediated nucleosome acetylation

  2018cites this paper
• Structural basis for activation of SAGA histone acetyltransferase Gcn5 by partner subunit Ada2

  2018influential citation
• The many lives of KATs — detectors, integrators and modulators of the cellular environment

  2018cites this paper
• Epigenetic Modulation Using Small Molecules - Targeting Histone Acetyltransferases in Disease.

  2017cites this paper
• Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine

  2017cites this paper
• Rôle de la chromatine dans la modulation de la réponse aux dommages à l’ADN en présence de stress réplicatif

  2017cites this paper
• Understanding Multiple Independent Functions of the Tip60 Acetyltransferase in Embryonic Development

  2017cites this paper
• Molecular Basis for Cohesin Acetylation by Establishment of Sister Chromatid Cohesion N-Acetyltransferase ESCO1*

  2016cites this paper
• Measuring specificity in multi-substrate/product systems as a tool to investigate selectivity in vivo.

  2016cites this paper
• Protein acetylation in metabolism — metabolites and cofactors

  2016cites this paper
• Histone acetyltransferases: challenges in targeting bi-substrate enzymes

  2016cites this paper
• Structural basis for acyl-group discrimination by human Gcn5L2

  2016cites this paper
• Competitive Inhibition of Lysine Acetyltransferase 2B by a Small Motif of the Adenoviral Oncoprotein E1A*

  2016cites this paper
• Activation of AMP-activated Protein Kinase by Metformin Induces Protein Acetylation in Prostate and Ovarian Cancer Cells*

  2016cites this paper
• Role of Histone-Modifying Enzymes and Their Complexes in Regulation of Chromatin Biology.

  2016cites this paper
• Mechanisms of histone lysine-modifying enzymes: A computational perspective on the role of the protein environment.

  2016cites this paper
• Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases

  2015cites this paper
• Competitive inhibition can linearize dose-response and generate a linear rectifier.

  2015cites this paper
• Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

  2015cites this paper
• Investigating the role of chromatin structure in modulating SAGA mediated nucleosomal acetylation

  2015cites this paper
• Defining the Orphan Functions of Lysine Acetyltransferases

  2015cites this paper
• Nucleosome competition reveals processive acetylation by the SAGA HAT module

  2015cites this paper
• Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

  2015cites this paper
• Investigations of sirtuin metabolism

  2015cites this paper
• Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors.

  2015cites this paper
• Acetyl coenzyme A: a central metabolite and second messenger.

  2015cites this paper
• Protein Lysine Acetylation by p300/CBP

  2015cites this paper
• Metabolic regulation of histone post-translational modifications.

  2015cites this paper
• SIRT3 mediates multi-tissue coupling for metabolic fuel switching.

  2015cites this paper
• The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization*

  2015cites this paper
• Structural and Biochemical Studies of Histone Acetyltransferases and Sirtuin Enzymes

  2015influential citation
• NOREPINEPHRINE-MEDIATED SYNAPTIC PLASTICITY: A NOVEL SIGNALING DIALOGUE BETWEEN RECEPTORS AND NUCLEUS

  2014cites this paper
• The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3*

  2014cites this paper
• Transcriptional and epigenetic regulation of Hebbian and non-Hebbian plasticity.

  2014cites this paper
• Nonenzymatic protein acylation as a carbon stress regulated by sirtuin deacylases.

  2014cites this paper
• Nucleosome acetylation sequencing to study the establishment of chromatin acetylation.

  2014cites this paper
• Acetyl Coenzyme A Synthetase Is Acetylated on Multiple Lysine Residues by a Protein Acetyltransferase with a Single Gcn5-Type N-Acetyltransferase (GNAT) Domain in Saccharopolyspora erythraea

  2014cites this paper
• Allosteric Regulation of a Protein Acetyltransferase in Micromonospora aurantiaca by the Amino Acids Cysteine and Arginine*

  2014cites this paper
• Protein Acetylation and Acetyl Coenzyme A Metabolism in Budding Yeast

  2014cites this paper
• The promise and failures of epigenetic therapies for cancer treatment.

  2014cites this paper
• Epigenetics of Neural Repair Following Spinal Cord Injury

  2013cites this paper
• Metabolic mechanisms of epigenetic regulation.

  2013cites this paper
• Identification and characterization of altered mitochondrial protein acetylation in Friedreich's Ataxia cardiomyopathy

  2013cites this paper
• Widespread and Enzyme-independent Nϵ-Acetylation and Nϵ-Succinylation of Proteins in the Chemical Conditions of the Mitochondrial Matrix*♦

  2013cites this paper
• Probing p300/CBP associated factor (PCAF)-dependent pathways with a small molecule inhibitor.

  2013cites this paper
• A study of coenzyme A metabolism and function in mammalian cells

  2013cites this paper
• Investigation of the Acetylation Mechanism by GCN5 Histone Acetyltransferase

  2012cites this paper
• Targeting the Histone Acetyl-Transferase, RTT109, for Novel Anti-Fungal Drug Development: A Dissertation

  2012influential citation
• Acetyl-CoA Carboxylase Regulates Global Histone Acetylation*♦

  2012cites this paper
• Novel functions of the MOZ double PHD finger domain

  2012cites this paper
• Processing mechanism and substrate selectivity of the core NuA4 histone acetyltransferase complex.

  2011cites this paper
• Probing the reaction coordinate of the p300/CBP histone acetyltransferase with bisubstrate analogs.

  2011cites this paper
• Biochemical and Thermodynamic Analyses of Salmonella enterica Pat, a Multidomain, Multimeric Nε-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism

  2011cites this paper
• Development of Inhibitors and Assay Methods for Histone Acetyltransferases

  2011cites this paper
• The Anaphase-Promoting Complex Interacts with Histone Modification Proteins and Chromatin Assembly Factors

  2011cites this paper
• KAT(ching) Metabolism by the Tail: Insight into the Links between Lysine Acetyltransferases and Metabolism

  2011cites this paper
• Reversible acetylation and inactivation of Mycobacterium tuberculosis acetyl-CoA synthetase is dependent on cAMP.

  2011cites this paper
• Kinetic mechanism of histone acetyltransferase and chaperone complex, Rtt109-Vps75 †

  2011cites this paper
• Pneumocystis carinii expresses an active Rtt109 histone acetyltransferase.

  2011cites this paper
• Histone acetylation modulation by small molecules: a chemical approach.

  2010cites this paper
• Lunasin Is Prevalent in Barley and Is Bioavailable and Bioactive in In Vivo and In Vitro Studies

  2010cites this paper
• Kinetic and Structural Insights into the Mechanism of AMPylation by VopS Fic Domain*

  2010cites this paper
• Kinetic mechanism of the Rtt109-Vps75 histone acetyltransferase-chaperone complex.

  2010cites this paper