Mammalian phenylalanine hydroxylase (PAH) is a multidomain homo-multimeric protein whose dysfunction causes the most common inborn error in amino acid metabolism, phenylketonuria (PKU), and milder forms of hyperphenylalaninemia. PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen. Phe allosterically activates PAH by binding to the regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein. Allosteric activation by Phe is accompanied by major conformational changes that move the N-terminal regulatory region away from the active site and stabilization of the tetrameric conformation.