Expression, Purification and Characterization of Carboxyltransferase Domain and Full‐Length Human Acetyl‐Coenzyme A Carboxylase 2

Acetyl‐Coenzyme A carboxylase (ACC) is a ~290 kDa protein which catalyzes the ATP dependent carboxylation of acetyl‐CoA into malonyl‐CoA, a key intermediate in the biosynthesis of fatty acids and a negative regulator of ‐oxidation in the mitochondria. Here we report the expression, purification and enzymatic characterization of both the carboxytransferase (CT) domain and full length human ACC2. His‐tagged CT and full‐ length human ACC2 have been successfully cloned, expressed using Sf21 insect cells, and purified to homogeneity. Full‐length hACC2 forms a multimeric complex with high specific activity and kinetic parameters very similar to the native ACC enzyme. In addition malonyl‐CoA exhibited competitive inhibition vs. acetyl‐CoA and non‐competitive inhibition vs. ATP in this protein. The hACC2 CT domain forms a dimer, and demonstrated a protein‐, time‐, and substrate‐dependent activity converting malonyl‐CoA to acetyl‐CoA. An inhibitor of full length human ACC2, bipiperidylcarbox‐amide, which binds to the putative biotin binding site, did not inhibit the CT‐domain mediated conversion of malonyl‐CoA to acetyl‐CoA, consistent with the absence of a requirement for biotin in the reaction. The generation of milligram quantities of active human ACC2 CT domain opens the door to structural determination of this important domain of ACC2. In addition the ability to produce both the full‐length protein and the CT domain will facilitate a more detailed characterization of ACC2 inhibitors.