Nucleotide-binding sites can enhance N -acylation of nearby protein lysine residues

Acyl-CoAs are reactive metabolites that can non-enzymatically S- acylate and N -acylate protein cysteine and lysine residues, respectively. N- acylation is irreversible and enhanced if a nearby cysteine residue undergoes an initial reversible S- acylation, as proximity leads to rapid S → N -transfer of the acyl moiety. We reasoned that protein-bound acyl-CoA could also facilitate S → N -transfer of acyl groups to proximal lysine residues. Furthermore, as CoA contains an ADP backbone this may extend beyond CoA-binding sites and include abundant Rossmann-fold motifs that bind the ADP moiety of NADH, NADPH, FADH and ATP. Here, we show that excess nucleotides decrease protein lysine N- acetylation in vitro. Furthermore, by generating modelled structures of proteins N -acetylated in mouse liver, we show that proximity to a nucleotide-binding site increases the risk of N- acetylation and identify where nucleotide binding could enhance N- acylation in vivo. Finally, using glutamate dehydrogenase as a case study, we observe increased in vitro lysine N- malonylation by malonyl-CoA near nucleotide-binding sites which overlaps with in vivo N- acetylation and N- succinylation. Furthermore, excess NADPH, GTP and ADP greatly diminish N- malonylation near their nucleotide-binding sites, but not at distant lysine residues. Thus, lysine N- acylation by acyl-CoAs is enhanced by nucleotide-binding sites and may contribute to higher stoichiometry protein N- acylation in vivo .