Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3

PLoS One. 2013;8(2):e54896. doi: 10.1371/journal.pone.0054896. Epub 2013 Feb 21.


Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36) and the catalytic efficiency (k(cat)/K(m)) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to -4 kcal/mol between K14 and K18, the highest and lowest measurable k(cat)/K(m). These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl Coenzyme A / metabolism
  • Acetylation
  • Amino Acid Sequence
  • Histones / chemistry
  • Histones / metabolism*
  • Humans
  • Kinetics
  • Lysine / metabolism
  • Mass Spectrometry
  • Molecular Sequence Data
  • Nucleosomes / metabolism
  • Peptides / chemistry
  • Peptides / metabolism
  • Reproducibility of Results
  • Staining and Labeling
  • Substrate Specificity
  • p300-CBP Transcription Factors / metabolism*


  • Histones
  • Nucleosomes
  • Peptides
  • Acetyl Coenzyme A
  • p300-CBP Transcription Factors
  • p300-CBP-associated factor
  • Lysine

Grant support

This project is funded, in part, under a grant with the Pennsylvania Department of Health. The Pennsylvania Department of Health specifically disclaims responsibility for any analysis, interpretations or conclusions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.