# Probing Enzymatic Acetylation Events in Real Time With NMR Spectroscopy: Insights Into Acyl‐Cofactor Dependent p300 Modification of Histone H4

## TL;DR

This paper introduces an NMR method to study enzyme-driven acetylation on histone proteins, revealing new insights into how different acyl cofactors affect the process.

## Contribution

The study expands NMR-based acylation detection to include propionylation and introduces a method for measuring relative reaction rates.

## Key findings

- NMR techniques were adapted to detect 13C-acetyl and 13C-propionyl modifications on histone H4.
- A continuous evaluation method was developed to extract relative rate constants from acyltransferase reactions.
- p300 KAT shows distinct site preferences and regulation depending on the acyl cofactor used.

## Abstract

Lysine acylation is a rapidly expanding class of post‐translational modifications with largely unexplored functional roles; the study of acylations beyond acetylation is especially impeded by limited methods for their preparation, detection, and characterization in vitro. We previously reported a nuclear magnetic resonance (NMR)‐based approach to monitor Nε‐lysine acetylation following Ada2/Gcn5‐catalyzed installation of a 13C‐acetyl probe on the histone H3 tail. Building on this foundation, here we expand those techniques by demonstrating the installation and 1H, 13C‐HSQC based NMR detection of both 13C‐acetyl and 13C‐propionyl probes on the histone H4 tail using a mutant p300 lysine acetyltransferase (KAT) enzyme with enhanced activity. Additionally, we introduce a continuous evaluation method for acyltransferase reaction data, enabling the extraction of relative rate constants—a technique inspired by our laboratory's recent work on NMR methyltransferase kinetics. This study demonstrates that our NMR‐based approach to assay enzymatic 13C‐acylation is adaptable, providing a versatile platform for investigating a range of acylations, KAT enzymes, and protein substrates. Notably, in the process of developing these methods, we observed that p300 KAT may display distinct modification site preferences and regulatory mechanisms depending on the acyl cofactor utilized, underscoring the method's potential to advance the emerging field of lysine acylation biochemistry.