BIOLOGICAL OXIDATION OF PHENYLALANINE BY NONHEME IRON

本研究的主要目的是阐明单核非血红素铁依赖金属酶苯丙氨酸羟化酶（PAH, phenylalanine 4-monooxygenase， E.C. 1.14.16.1）的化学和物理性质。多环芳烃是一种多功能氧化酶，其活性位点亚铁中心在双氧和还原的辅助因子四氢蝶呤存在下催化l -苯丙氨酸（L-phe）羟基化为l -酪氨酸（L-tyr）。PAH基因的遗传缺陷导致PAH活性显著降低，导致常染色体隐性人类遗传疾病，经典苯丙酮尿症（PKU）。这种疾病的特点是血清l -苯丙氨酸浓度增加和苯丙氨酸代谢产物的异常积累，这些代谢产物被认为会导致中枢神经系统髓鞘形成缺陷，导致出生后脑损伤和严重的智力迟钝。PKU是氨基酸代谢中最常见的先天性错误，具有重要的临床意义；大约每50人中就有1人携带这种疾病特征，白种人的平均发病率约为万分之一。早期发现和严格的饮食管理可以显著减少未经治疗的PKU的神经缺陷和智力迟钝特征。该项目的具体目标之一是根据其结构和化学含义，识别和解释活性位点铁环境的变化作为必要的变构活化过程的功能，以及底物和辅因子的结合。先进的方法，包括MCD， XAS, Mssbauer， ESEEM和ENDOR光谱，将用于探测多种生理相关酶状态的活性位点结构，所有这些都可以以均相形式生成。将对天然的同四聚体多环芳烃的活性位点特征与二聚体/单体形式的酶进行比较；截断二聚体形式的多环芳烃支持混杂氧化化学，即底物可以被氧化，而野生型全长多环芳烃只有效地接受l -苯二酚作为底物。其他目标是比较野生型多环芳烃与选定的PKU诱导错意突变，作为对多环芳烃机制的进一步探索，并启动旨在定义高苯丙氨酸血症疾病化学基础的研究。最后，利用双混合停流UV/vis和荧光实验来探测底物催化氧化前铁活性位点的强制还原。动力学和机械实验将进行，以确定控制和调节铁还原的因素。

The major objective of this research is to elucidate the chemical and physical properties of the mononuclear non-heme iron dependent metalloenzyme, phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase, E.C. 1.14.16.1). PAH is a mixed-function oxidase whose active site ferrous iron center catalyzes the hydroxylation of L-phenylalanine (L-phe) to L-tyrosine (L-tyr) in the presence of dioxygen and the reduced cofactor, tetrahydropterin. Genetic defects in the PAH gene that induce a significant decrease in PAH activity lead to the autosomal recessive human genetic disorder, classic phenylketonuria (PKU). This disorder is characterized by an increase in serum L-phe concentrations and the abnormal accumulation of phenylalanine-based metabolic products that are thought to cause defective myelination of the central nervous system, leading to postnatal brain damage and severe mental retardation. PKU is the most common inborn error in amino acid metabolism that is of clinical importance; approximately 1 in 50 individuals carry the disease trait with an average incidence of about 1 in 10,000 for Caucasians. Early detection and strict dietary management have significantly reduced the neurological defects and mental retardation characteristics of untreated PKU. Among the specific aims of this project is to identify and interpret, in terms of their structural and chemical implications, changes in the active site iron environment as a function of the requisite allosteric activation process, and the binding of substrate(s) and cofactor. Advanced methodologies, including MCD, XAS, Mssbauer, ESEEM, and ENDOR spectroscopies, will be utilized to probe the active site structures of multiple physiologically relevant enzyme states, all of which can be generated in homogeneous forms. A comparison of the active site characteristics of native, homotetrameric PAH with dimeric/monomeric forms of the enzyme will be performed; truncated dimeric forms of PAH support promiscuous oxidation chemistry in terms of which substrates can be oxidized versus wildtype full length PAH, which only efficiently accepts L-phe as substrate. Additional objectives are to compare wildtype PAH with selected PKU inducing missense mutations as a further probe of PAH mechanism and to initiate studies designed to defining the chemical basis for hyperphenylalaninemic disorders. Finally, the use of double mixing stopped-flow UV/vis and florescence experiments will be utilized to probe the compulsory reduction of the ferric active site prior to catalytic oxidation of substrate. Kinetic and mechanistic experiments will be performed to identify the factors that control and regulate iron reduction.