Mechanisms of Aromatic Amino Acid Hydroxylases

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Phenylalanine hydroxylase (PheH), tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (TrpH) make up the family of tetrahydropterin dependent hydroxylases. These enzymes catalyze the iron-dependent aromatic hydroxylation of their respective substrates. PheH in the liver catalyzes the hydroxylation of phenylalanine to tyrosine. Mutations in PheH result in the inherited disease phenylketonuria. TyrH in the brain and adrenal gland catalyzes the hydroxylation of tyrosine to DOPA, the first step in the biosynthesis of the catecholamine neurotransmitters. Mutations in TyrH have been associated with Parkinson's disease. TrpH in the brain catalyzes the hydroxylation of tryptophan to 5-hydroxytryptophan,the first step in the biosynthesis of the neurotransmitter serotonin. Low levels of serotonin have been associated with depression, violence, and alcoholism. The chemical mechanism of TyrH has been studied extensively, and this proposal will expandon the previous work. The mechanism of oxygen activation by PheH and TrpH will be studied using 18O kinetic isotope effects.The reactivity of the hydroxylating intermediate in all three enzymes will be studied by measuring deuterium isotope effects on benzylic and aromatic hydroxylation. Rapid quench EPR and Mossbauer spectroscopy will be used for spectroscopic characterization of the proposed hydroxylating intermediate.