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）：二胺氧化酶（DAO）活性在分离静止和快速分裂细胞的边界中升高，其中通过这些酶将分子氧和生物胺转化为氨基醛和过氧化氢可能在正常和癌性组织的细胞分裂调节中起重要作用。对这种酶的生化研究将有助于阐明正常和疾病状态下细胞对多胺浓度变化反应的分子机制。在这里，我们建议1）开发和优化从牛肝脏和肾脏中的二胺氧化酶的纯化方案，并评估这些异构体的底物特异性2）确定锰在配体结合和催化机制中的作用，3）表征DAO的肝素和核酸结合相互作用及其与这些生物聚合物相关的催化活性的调节。将比较纯化的同工酶的一级结构、糖基化、辅因子含量、比活性和底物特异性。 纯化的酶的催化反应机制将通过结合配体结合，稳态，和预稳态动力学测量进行研究。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，停流和连续流测量的组合进行研究。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特定影响来建立。通过停流光谱法观察氧化和还原半反应动力学的变化，确定核酸结合对各个反应步骤的影响。多胺在DAO-核酸结合相互作用中的作用和DAO与多胺的氧化动力学将被研究。 二胺浓度升高和二胺氧化酶活性增加会产生显著浓度的过氧化氢和氨基醛，这可能导致氧化损伤和易感蛋白质和核酸的反应基团之间分子间和分子内交联的形成。将通过毛细管电泳和质谱法筛选从规定组成的体外反应中回收的产物的DAO诱导的分子修饰。