Phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is an enzyme that catalyzes the hydroxylation of the aromatic side-chain of phenylalanine to generate tyrosine. PAH is one of three members of the biopterin-dependent aromatic amino acid hydroxylases, a class of monooxygenase that uses tetrahydrobiopterin (BH4, a pteridine cofactor) and a non-heme iron for catalysis. During the reaction, molecular oxygen is heterolytically cleaved with sequential incorporation of one oxygen atom into BH4 and phenylalanine substrate. In humans, mutations in its encoding gene, PAH, can lead to the metabolic disorder phenylketonuria. Deficiency in PAH activity due to mutations in PAH causes hyperphenylalaninemia (HPA). Since phenylketonuria can cause irreversible damage, it is imperative that deficiencies in the phenylalanine hydroxylase are determined early on in development. Originally, this was done using a bacterial inhibition assay known as the Guthrie Test. Now, PKU is part of newborn screening in many countries, and elevated phenylalanine levels are identified shortly after birth by measurement with tandem mass spectrometry. Placing the individual on a low phenylalanine, high tyrosine diet can help prevent any long-term damage to their development.