Mechanisms of Aromatic Amino Acid Hydroxylases

描述（由申请人提供）：苯丙氨酸羟化酶（PheH），酪氨酸羟化酶（TyrH）和色氨酸羟化酶（TrpH）构成四氢蝶呤依赖羟化酶家族。这些酶催化各自底物的铁依赖性芳香羟基化。肝脏中的PheH催化苯丙氨酸羟基化为酪氨酸。PheH突变导致遗传性疾病苯丙酮尿症。大脑和肾上腺中的TyrH催化酪氨酸羟基化为多巴，这是儿茶酚胺神经递质生物合成的第一步。TyrH突变与帕金森病有关。大脑中的TrpH催化色氨酸羟基化为5-羟色氨酸，这是神经递质血清素生物合成的第一步。低水平的血清素与抑郁、暴力和酗酒有关。TyrH的化学机理已经得到了广泛的研究，本论文的提出是对前人研究的补充。利用18O动力学同位素效应研究PheH和TrpH对氧的活化机理。羟基化中间体在所有三种酶中的反应性将通过测量氘同位素对苯基和芳香族羟基化的影响来研究。快速猝灭EPR和穆斯堡尔光谱将用于所提出的羟基化中间体的光谱表征。

DESCRIPTION (provided by applicant): 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 expand on 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.