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: 6520232
• 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/6520232
• 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金属酶苯丙氨酸羟基酶(PAH，苯丙氨酸4-单加氧酶，E.C.1.14.16.1)的化学和物理性质。多环芳烃是一种功能齐全的酶，其活性中心亚铁中心在氧气和还原辅因子四氢蝶呤存在下，催化L-苯丙氨酸(L-苯丙氨酸)羟化成L-酪氨酸(L-酪氨酸)。PAH基因的遗传缺陷导致PAH活性显著降低，导致人类常染色体隐性遗传性疾病，经典的苯丙酮尿症(PKU)。这种疾病的特征是血清L-苯丙氨酸浓度升高和苯丙氨酸代谢产物的异常积聚，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低下。PKU是最常见的具有临床重要性的氨基酸代谢先天错误；大约每50人中就有1人携带这种疾病特征，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著减少了未经治疗的PKU患者的神经功能缺陷和智力低下特征。该项目的具体目标之一是从结构和化学影响的角度确定和解释活性中心铁环境的变化，这是必要的变构激活过程以及底物(S)和辅因子结合的结果。先进的方法，包括MCD，XAS，穆斯堡尔，ESEEM和Endor光谱学，将被用来探测多种生理相关酶状态的活性中心结构，所有这些都可以以均一的形式产生。我们将比较天然的同源四聚体多环芳烃和酶的二聚/单体形式的活性中心特征；截短的多环芳烃二聚体形式支持底物可以被氧化的混杂氧化化学，而野生型全长多环芳烃只有效地接受L-Phe作为底物。其他目标是比较野生型PAH和选择的PKU诱导的错义突变，以进一步探讨PAH的机制，并启动旨在确定高苯丙氨酸血症疾病的化学基础的研究。最后，将利用双混合停流UV/Vis和荧光实验来探索在底物催化氧化之前铁的活性中心的强制还原。将进行动力学和力学实验，以确定控制和调节铁还原的因素。