Related Experiment Video
Updated: May 11, 2026
07:37
Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening
Published on: May 13, 2020
2.0K
A fluorescent CPM-based in vitro acetylation assay: A tool for assessing N-terminal acetyltransferase activity and
Franziska Mueller1, Matthias Bischoff2, Sascha Gentz3
1Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Methods in Enzymology
|August 31, 2025
Summary
Researchers developed a new fluorescence assay to measure N-terminal acetyltransferase (NAT) activity. This method enables high-throughput screening of NAT enzymes and potential drug inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- N-terminal acetylation is a crucial co-translational modification in eukaryotes, impacting approximately 80% of human cytosolic proteins.
- This modification, catalyzed by N-terminal acetyltransferases (NATs), regulates vital protein functions including stability, localization, and interactions.
- Existing high-throughput, non-radioactive methods for studying NAT activity are limited, hindering comprehensive research.
Purpose of the Study:
- To develop and optimize a novel, sensitive, and high-throughput fluorescence-based assay for monitoring N-terminal acetyltransferase (NAT) activity.
- To establish a robust workflow for utilizing this assay with human NAT enzymes (NatA and NatB) for kinetic measurements and inhibitor screening.
- To validate the assay's performance in various well formats and discuss potential interferences and validation strategies.
Main Methods:
- A fluorescence-based assay utilizing 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) was optimized to detect free coenzyme A released during acetyl transfer.
- A comprehensive workflow was established, encompassing enzyme purification, reaction setup, and real-time assay readout for human NatA and NatB.
- The assay was miniaturized to 384- and 1536-well formats and validated using a bisubstrate inhibitor, with optimization of enzyme/substrate concentrations and time-course analysis.
Main Results:
- The optimized CPM assay demonstrated high sensitivity and a linear correlation between CPM fluorescence and released coenzyme A.
- The assay enables accurate kinetic measurements and inhibitor testing in a 96-well format, with successful miniaturization to 384- and 1536-well formats.
- Optimization parameters and potential assay interferences were detailed, alongside recommendations for orthogonal validation to ensure inhibitor specificity.
Conclusions:
- The developed fluorescence assay provides a sensitive, high-throughput, and non-radioactive method for studying NAT activity.
- This assay facilitates accurate kinetic analysis and inhibitor screening for NAT enzymes, supporting drug discovery efforts.
- The established workflow and optimization guidelines offer a valuable tool for researchers investigating N-terminal acetylation.
