Ligand-Binding in the Reaction Mechanism of DAO

DAO 反应机制中的配体结合

• 批准号: 6838246
• 负责人: George T. Gassner
• 金额: $ 7.5万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6838246
• 关键词: DNA damage; amine oxidase (copper); animal tissue; atomic absorption spectrometry; capillary electrophoresis; catalyst; chemical binding; enzyme activity; enzyme substrate; gel filtration chromatography; heparin; high performance liquid chromatography; ion exchange chromatography; isozymes; ligands; manganese; mass spectrometry; nucleoproteins; oxidative stress; protein purification; stop flow technique

DESCRIPTION (provided by applicant): Diamine oxidase (DAO) activities are elevated in boundaries separating quiescent and rapidly dividing cells where the transformation of molecular oxygen and biogenic amines to aminoaldehydes and hydrogen peroxide by these enzymes may play a significant role in the regulation of cell division in normal and cancerous tissues. Biochemical studies of this enzyme will help to elucidate the molecular mechanism of cellular response to changes in polyamine concentration in normal and disease states. Here we propose to 1) develop and optimize purification schemes for diamine oxidase from bovine liver and kidney and evaluate the substrate specificities of these isoforms 2) identify the role of manganese in ligand-binding and the catalytic mechanism, 3) characterize the heparin, and nucleic acid-binding interactions of DAO and the modulation of its catalytic activity associated with these biopolymers. The primary structure, glycosylation, cofactor content, specific activity, and substrate specificities of the purified isozymes will be compared. Catalytic reaction mechanisms of the purified enzymes will be studied through a combination of ligand binding, steady state, and pre-steady state kinetic measurements. DNA, RNA, heparin, and metal binding mechanisms, as well as the polynucleotide sequence binding specificity of these isozymes will be investigated by using a combination of titrametric, stopped-flow and continuous flow measurements. The role of polynucleotide, metal, and heparin-binding in catalysis will be established through the measurement of ligand-specific effects on steady-state kinetic parameters and reaction product distributions. The influence of nucleic acid binding on individual reaction steps will be established by observing changes in the kinetics of the oxidative and reductive half reactions by stopped-flow spectroscopy. The role of polyamines in the DAO-nucleic acid binding interaction and the kinetics of oxidation of DAO with polyamines will be investigated. Elevated concentrations of diamines and increased diamine oxidase activity generates significant concentrations of hydrogen peroxide and aminoaldehydes, which may lead to oxidative damage and the formation of inter- and intramolecular cross-links between reactive groups of susceptible proteins and nucleic acids. Products recovered from in vitro reactions of defined composition will be screened for DAO-induced molecular modifications by capillary electrophoresis and mass spectrometry.

描述（由申请人提供）：分离静态和快速分裂细胞的边界中二氨酸氧化酶（DAO）活性升高，在这些细胞中，这些酶对分子氧和生物胺对氨基醛和过氧化氢的转化可能在正常和癌症组织的细胞分裂中起重要作用。该酶的生化研究将有助于阐明正常和疾病状态中多胺浓度变化的细胞反应的分子机制。在这里，我们提出1）开发和优化牛肝和肾脏二氨酸氧化酶的纯化方案，并评估它们的同工型的底物特异性2）确定锰在配体结合和催化机制中的作用，3）与肝素结合的表征和核酸结合的作用与核酸的相互作用和核酸结合的作用相关，并与核酸的相互作用相关。将比较纯化同工酶的主要结构，糖基化，辅因子含量，特定活性和底物特异性。 纯化酶的催化反应机制将通过配体结合，稳态和稳态态动力学测量的结合进行研究。 DNA，RNA，肝素和金属结合机制，以及这些同工酶的多核苷酸序列结合特异性将通过使用滴定，停止流量和连续流量测量的组合来研究。多核苷酸，金属和肝素结合在催化中的作用将通过测量配体对稳态动力学参数和反应产物分布的测量来确定。核酸结合对单个反应步骤的影响将通过观察到通过停止流量光谱法观察氧化和还原性半反应动力学的变化。多胺在Dao核酸结合相互作用和DAO与多胺的氧化动力学中的作用。 氨基氨酸的浓度升高和二氨酸氧化酶活性的增加会产生大浓度的过氧化氢和氨基醛，这可能导致氧化损伤，并形成可易感蛋白质和核酸的反应性群体之间的分子间和分子内交联。从定义组成的体外反应中回收的产物将通过毛细管电泳和质谱法进行DAO诱导的分子修饰。