BIOLOGICAL OXIDATION OF PHENYLALANINE BY NONHEME IRON

非血红素铁对苯丙氨酸的生物氧化

• 批准号: 6090321
• 负责人: John P. Caradonna
• 金额: $ 19.56万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6090321
• 关键词: Mossbauer spectrometry; X ray crystallography; active sites; affinity chromatography; animal tissue; circular magnetic dichroism; colorimetry; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; enzyme structure; gene mutation; high performance liquid chromatography; human tissue; iron sulfur protein; metalloenzyme; nucleic acid sequence; oxidation; phenylalanine 4 monooxygenase; phenylketonurias; polymerase chain reaction; protein binding; site directed mutagenesis; stop flow technique; ultraviolet spectrometry

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.

本研究的主要目的是阐明单核非血红素铁依赖性金属酶苯丙氨酸羟化酶（PAH，Phenylalanine 4-monooxygenase，E.C.）的化学和物理性质。1.14.16.1）。 PAH是一种多功能氧化酶，其活性位点亚铁中心在分子氧和还原辅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基因中的遗传缺陷可导致PAH活性显著降低，从而导致常染色体隐性遗传性人类遗传疾病，经典型苯丙酮尿症（PKU）。 这种疾病的特征在于血清L-phe浓度的增加和苯丙氨酸基代谢产物的异常积累，苯丙氨酸基代谢产物被认为引起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是具有临床重要性的氨基酸代谢中最常见的先天性缺陷;大约50个个体中有1个携带该疾病特征，高加索人的平均发病率约为1/10，000。 早期发现和严格的饮食管理显着减少了未经治疗的PKU的神经缺陷和智力低下特征。该项目的具体目标之一是确定和解释，在其结构和化学影响方面，活性位点铁环境的变化作为必要的变构活化过程的函数，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，将被用来探测多种生理相关的酶状态的活性位点结构，所有这些都可以以均匀的形式产生。 将进行天然同源四聚体PAH与酶的二聚体/单体形式的活性位点特征的比较; PAH的截短二聚体形式支持混杂的氧化化学，就其底物可以被氧化而言，野生型全长PAH仅有效地接受L-phe作为底物。 其他目的是比较野生型PAH与选择的PKU诱导错义突变作为PAH机制的进一步探针，并启动旨在定义高苯丙氨酸血症疾病的化学基础的研究。 最后，使用双混合停流UV/维斯和荧光实验将被用来探测强制还原的铁活性位点之前，催化氧化的基板。将进行动力学和机械实验，以确定控制和调节铁还原的因素。