Site-Specific incorporation of lysine acetyl-methylation into proteins.

Lysine acetyl-methylation (Kam) is a novel protein post-translational modification (PTM) recently identified on histone H4, yet its biological functions on non-histone proteins remain largely unexplored. Here, we engineered an orthogonal Methanosarcina barkeri pyrrolysyl-tRNA synthetase (MbPylRS) variant and tRNACUAPyl pair for site-specific Kam incorporation into proteins in mammalian cells. Using this system, we identified endogenous Kam sites at K28 of adenylate kinase 2 (AK2) and K207 of pyruvate kinase M2 (PKM2) through proteomic analysis. Functional characterization revealed that Kam at these evolutionarily conserved residues significantly attenuated enzymatic activity. These findings demonstrate that Kam serves as a regulatory mechanism for non-histone proteins, and the ability to generate proteins harboring Kam at defined sites provides a valuable approach for investigating the biological roles of this newly identified PTM.