Regulation of Phenylalanine Hydroxylase

 描述(申请人提供)：苯丙氨酸羟基酶(PheH)催化膳食苯丙氨酸羟基化为酪氨酸。缺乏功能性PheH会导致代谢性疾病苯丙酮尿症(PKU)。PheH是三种芳香族氨基酸羟基酶之一，另外两种是酪氨酸羟基酶和色氨酸羟基酶。对苯丙氨酸的调节受到严格控制，因此只消耗过量的苯丙氨酸。该酶通过苯丙氨酸的激活、四氢生物蝶呤(BH4)的抑制和Ser16的磷酸化来调节。这项提案的目的是了解这些调节机制如何共同作用来控制苯丙氨酸的消费。我建议通过1)确定苯丙氨酸结合如何引起PheH的激活和2)确定蝶呤如何抑制PheH的激活来完成这项工作。苯丙氨酸活化的动力学参数为 使用野生型PheH、磷酸化PheH和∆24PheH，采用停流荧光光谱法测定。化学计量比和苯丙氨酸与这些酶结合的能量将通过等温量热法(ITC)确定。将确定两个PKU突变体I65T和R68S对PheH激活的影响。蝶呤结合的动力学参数(BH4、6MPH4和空气稳定的5-去氮-6-甲基四氢蝶呤)将用停流吸收光谱测定。这些实验将在E286A和S23a突变体上重复进行。ITC将用于测定蝶呤结合的化学计量学和能量学，并分析两个突变体活性部位的蝶呤结合。5-去氮-6-甲基四氢蝶呤和BH4将被用来确定蝶呤对PheH激活的影响。这一提议是创新的，因为它具有新颖的激活机制。这些研究将为理解PheH的调节机制和PKU引起的突变的作用提供重要的影响。

 DESCRIPTION (provided by applicant): Phenylalanine hydroxylase (PheH) catalyzes the hydroxylation of dietary phenylalanine to tyrosine. Lack of a functional PheH results in the metabolic disease phenylketonuria (PKU). PheH is one of three aromatic amino acid hydroxylases; the other two are tyrosine hydroxylase and tryptophan hydroxylase. Regulation of PheH is tightly controlled so that only excess phenylalanine is consumed. The enzyme is regulated by activation by phenylalanine, inhibition by tetrahydrobiopterin (BH4), and phosphorylation at Ser16. The goal of this proposal is to understand how these regulatory mechanisms work together to control phenylalanine consumption. I propose to do this by 1) determining how phenylalanine binding causes the activation of PheH and 2) determining how pterin inhibits the activation of PheH. The kinetic parameters for phenylalanine activation will be determined by stopped-flow fluorescence spectroscopy, using the wild-type PheH, phosphorylated PheH and ∆24 PheH. The stoichiometry and the energetics of phenylalanine-binding to these enzymes will determined by isothermal calorimetry (ITC). The effect of two PKU mutants, I65T and R68S, on activation of PheH will be determined. The kinetic parameters of pterin binding (BH4, 6MPH4, and the air-stable 5-deaza-6-methyltetrahdropterin) will be determined by stopped-flow absorbance spectroscopy. These experiments will be repeated with the E286A and S23A mutants. ITC will used to determine the stoichiometry and energetics of pterin binding and to analyze binding of pterin in the active site in the two mutants. 5-Deaza-6-methyltetrahdropterin and BH4 will be used to determine the effects of pterin on activation of PheH. This proposal is innovative because of the novel mechanism of activation. These studies will provide significant impact on the understanding of the mechanism for the regulation of PheH and the roles of PKU-causing mutations.