Adenosine triphosphate can act as a determinant of lysine acetylation of non-native and native substrates.

The protein lysine acetylation includes acetyl-CoA (AcCoA) or acetyl phosphate (AcP)-mediated nonenzymatic acetylation, and enzymatic acetylation. It is widespread in the proteomes but the acetylation levels of most sites are very low. A thorough understanding of the determinants of low acetylation levels is highly important for elucidating the physiological relevance of lysine acetylation. In this study, we constructed a non-native substrate library containing 24 synthesized polypeptides, and we showed that ATP could inhibit the AcCoA-mediated nonenzymatic acetylation of these polypeptides through LC-MS/MS analysis. The acetyltransferase PatZ could acetylated these non-native substrates, and the PatZ-catalyzed acetylation of the polypeptides was also inhibited by ATP. Furthermore, the Western blot showed that ATP also inhibited the nonenzymatic (AcCoA or AcP-mediated) and enzymatic (PatZ-catalyzed) acetylation of acetyl-CoA synthetase Acs, which is a native substrate for acetylation. ATP can also inhibit the autoacetylation of acetyltransferase PatZ. Besides, both ADP and AMP could enhance the AcP-mediated acetylation of Acs, but ADP slightly inhibited the AcCoA-mediated acetylation of Acs. However, both ADP and AMP had no evident inhibition on the PatZ-catalyzed acetylation of Acs. Based on these results, we proposed that ATP can act as an inhibitor of acetylation, and it may regulate the function of PatZ by inhibiting its autoacetylation and compensate for the function of deacetylase CobB.