Protein lysine acetylation is a reversible posttranslational modification that is catalyzed by a group of enzymes that are collectively referred to as lysine (K) acetyltransferases (KATs). These enzymes catalyze the transfer of the acetyl group from acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine amino acid. Protein lysine acetylation plays a critical role in the regulation of important cellular processes and it is therefore paramount that we understand the catalytic mechanisms of these enzymes. While there is a variety of methods that have been developed to analyze the enzymatic properties of KATs, majority of the proposed methods have considerable limitations. We describe here a reversed phase HPLC based method that monitors substrate consumption and product formation simultaneously. This method is highly reproducible and optimally suited for the determination of accurate kinetic parameters of KATs.
Keywords: In vitro protein lysine acetylation; Lysine acetyltransferases (KATs); Reversed phase high performance liquid chromatograph (HPLC).