Studies of ascorbate-dependent, non-heme iron enzyme ACC oxidase

抗坏血酸依赖性非血红素铁酶 ACC 氧化酶的研究

• 批准号: 7273395
• 负责人: LIVIU M. MIRICA
• 金额: $ 4.96万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7273395
• 关键词: Active Sites; Agriculture; Anabolism; Area; Binding; Carboxylic Acids; Consumption; Coupling; Dioxygen; Enzymes; Ethylene; Ethylenes; Fruit; Germination; Goals; Heme Iron; Human; Iron; Isotopes; Kinetics; L-Ascorbate oxidase; Label; Measurement; Medical; Methods; Mononuclear; Nature; Oxidants; Oxidases; Oxygen; Pharmacologic Substance; Plant Growth Regulators; Plants; Process; Radioactive; Range; Reaction; Reducing Agents; Refrigeration; Research; Role; Series; Time; analog; ascorbate; base; cofactor; design; inhibitor/antagonist; nutrition; oxidation; plant growth/development; research study; senescence; wasting

DESCRIPTION (provided by applicant): Oxygen-activating enzymes with mononuclear non-heme iron active sites participate in many metabolically important reactions that have tremendous medical, pharmaceutical, and environmental significance, catalyzing a wide range of reactions involving dioxygen (monooxygenation, oxidation, etc.). The present proposal aims to obtain more information about the mechanism by which such iron enzymes activate dioxygen and how cofactors and substrates interact in the active site to allow for catalytic activity. Studies will focus on 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO), a unique ascorbate-dependent, non- heme iron enzyme that catalyzes the last step in the biosynthesis of plant hormone ethylene, important in many aspects of plant growth and development, including germination, fruit ripening, and senescence. The specific aims of this proposal are 1) studying the mode of oxygen activation by the iron center in ACCO and the nature of the active oxidant using 18O kinetic isotope effects and pre-steady state kinetics and 2) deciphering the substrate breakdown mechanism by using alternate substrates containing radioactive labels. A range of studies are planned that include the measurement of 18O kinetic isotope effects to determine the order of reductant binding and the role of ascorbate in the reaction, and the characterization of intermediates that accumulate during catalytic turnover by stopped-flow methods. Radioactively labeled substrate analogs will be used in enzyme inactivation studies which could be potentially used for designing mechanism-based inhibitors that could help reduce agricultural waste and sustain healthy nutrition. The ability to control the ethylene synthesis processes in a time-dependent manner may aid in decreasing major losses of agricultural waste due to over-ripening. Potential impact on human healty is significant, particularly where long distance transport of produce is needed or storage occurs in areas that lack sufficient refrigeration.

描述（由申请人提供）：具有单核非血红素铁活性位点的氧活化酶参与许多具有巨大医学、制药和环境意义的代谢重要反应，催化涉及双氧的广泛反应（单氧合、氧化等） .)。本提案旨在获得有关此类铁酶激活分子氧的机制以及辅因子和底物如何在活性位点相互作用以实现催化活性的更多信息。研究将集中于 1-氨基环丙烷-1-羧酸氧化酶 (ACCO)，这是一种独特的抗坏血酸依赖性、非血红素铁酶，可催化植物激素乙烯生物合成的最后一步，对植物生长和发育的许多方面都很重要，包括发芽、果实成熟和衰老。该提案的具体目标是 1) 利用 18O 动力学同位素效应和前稳态动力学研究 ACCO 中铁中心的氧活化模式和活性氧化剂的性质，以及 2) 通过使用替代方法破译底物分解机制含有放射性标记的底物。计划进行一系列研究，包括测量 18O 动力学同位素效应以确定还原剂结合的顺序和抗坏血酸在反应中的作用，以及通过停流方法表征催化周转过程中积累的中间体。放射性标记的底物类似物将用于酶失活研究，这可能用于设计基于机制的抑制剂，有助于减少农业废物并维持健康营养。以时间依赖性方式控制乙烯合成过程的能力可能有助于减少由于过度成熟而造成的农业废物的重大损失。对人类健康的潜在影响是巨大的，特别是在需要长途运输农产品或在缺乏足够冷藏的地区储存的情况下。