Understanding the role of lysine acetylation in the regulation of Pyruvate Kinase M2 using the genetic code expansion method

Pyruvate kinase M2 (PKM2) is one of four isoforms of the glycolytic pyruvate kinase enzyme and can be found mostly in differentiated tissues and human cancer cells. PKM2 catalyzed the last step within glycolysis which is the convert of phosphoenolpyruvate to pyruvate and production of one ATP molecule. PKM2 is highly regulated by post-translation modification such as phosphorylation, Oxidation, ubiquitination and acetylation that can change PKM2 activity to a nonglycolytic enzyme and affect its oligomeric state. Acetylation refers to a process which Acetyl group is adding on the N-terminus or on a specific lysine within the protein sequence. In order to understand how acetylated lysine (AcK) affects PKM2 activity and structure, 9 suspected lysines were chosen. Till now, the effect of AcK was studied by creating a site mutation to glutamine which is different from AcK by its charge and structure. To overcome this problem, we are using the genetic code expansion method which can modify the genetic code and encode to an unnaturally-amino acid by using TAG stop codon, a specific tRNA, a tRNA synthetase and an unnatural amino acid. Using the genetic code expansion and the unnatural amino acid, AcK, all the nine different PKM2 variants, each of them has an AcK in a specific position, were expressed and purified. The WT and the PKM2 variants were used in PK activity assay and in protein structural studies. In parallel, all those PKM2 variants were expressed in HEK293T cell in order to check their activity in a cell lysate.