Activity-Based Substrate Profiling for Gcn5-Related N-Acetyltransferases:  The Use of Chloroacetyl-Coenzyme A To Identify Protein Substrates

The Gcn5-related N-acetyltransferases (GNAT) comprise one of the largest enzyme superfamilies, with over 10 000 known members represented in all kingdoms of life. ChloroacetylCoenzymeA was prepared and demonstrated to be a substrate for several GNAT members. ChloroacetylCoA (ClAcCoA) is used by the Hat1 histone acetyltransferase to correctly acetylate histone H4 in a mixture of histone proteins. Chloroacetylation can be assessed by the subsequent reaction of the chloroacetylated product with thiol-containing compounds, including those with fluorescent or affinity (His8) tags. The bacterial RimL N-acetyltransferase also uses ClAcCoA to chloroacetyl the α-amino group of its cognate substrate, the ribosomal L12 protein, and this reaction can be observed in crude extracts. ChloroacetylCoA is a reagent that can be used to identify the unknown substrate(s) for this large family of functionally uncharacterized enzymes.