KDAC8 substrate specificity quantified by a biologically relevant, label-free deacetylation assay

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Protein acetylation is a crucial post-translational modification regulating numerous biological processes.
• Lysine deacetylase (KDAC) enzymes reverse acetylation, but their substrate recognition mechanisms remain poorly understood.
• Existing methods for studying KDAC activity lack biological relevance and are not ideal for identifying enzyme-substrate pairs.

Purpose of the Study:

• To develop a more biologically relevant assay for probing KDAC substrate specificity.
• To investigate the substrate preferences of KDAC8 and other KDACs.
• To elucidate the role of surrounding residues in KDAC-substrate interactions.

Main Methods:

• Development of a novel fluorescamine-based assay for monitoring label-free protein deacetylation.
• Evaluation of KDAC8 and other KDACs' activity against peptides from known acetylated proteins.
• Analysis of steady-state kinetics to determine the impact of substrate sequence on binding and catalysis.

Main Results:

• The new assay demonstrated KDAC8's preference for specific peptide substrates, highlighting the importance of the amino acid sequence around acetylated lysine.
• Kinetic analysis indicated that substrate sequence independently influences both binding affinity and catalytic rate.
• Results validated potential KDAC8 substrates identified in cell-based experiments, contrasting with previous less relevant screening methods.

Conclusions:

• The developed fluorescamine assay provides a biologically relevant tool for studying KDAC substrate specificity.
• KDAC8 exhibits distinct substrate preferences, governed by the sequence context of the acetylated lysine residue.
• Integrating this assay with mass spectrometry and cell-based approaches will facilitate the identification of specific KDAC-substrate interactions and their biological implications.