Pyruvate carboxylase is an enzyme of gluconeogenesis. It catalyzes formation of a carboxyl group on pyruvate (using CO2) to make oxaloacetate.

Pyruvate + HCO3^- + ATP <=> Oxaloacetate + ADP + Pi + H⁺ 

Pyruvate carboxylase is activated allosterically by acetyl-CoA. The enzyme is a tetrameric protein carrying four molecules of biotin, each bound covalently through an amide bond involving the -amino group of a lysine residue. In animals the reaction catalyzed by pyruvate carboxylase is the most important anaplerotic reaction, particularly in liver and kidney. Pyruvate carboxylase is the only enzyme of gluconeogenesis in the mitochondria, requiring pyruvate and oxaloacetate to be transported across the mitochondrial membrane for gluconeogenesis to occur.