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激活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（单氧，氧化等）的广泛反应。目前的建议旨在获得更多的信息，通过这种铁酶激活双氧的机制，以及辅因子和底物如何在活性位点相互作用，以允许催化活性。研究将集中在1-氨基环丙烷-1-羧酸氧化酶（ACCO）上，这是一种独特的抗坏血酸依赖的非血红素铁酶，催化植物激素乙烯生物合成的最后一步，在植物生长和发育的许多方面，包括发芽，果实成熟和衰老都很重要。本课题的具体目标是：1)利用18O动力学同位素效应和预稳态动力学研究ACCO中铁中心的氧活化模式和活性氧化剂的性质；2)利用含有放射性标记的替代底物来破译底物的分解机制。一系列的研究计划包括测量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，以及通过停止流动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，这可能潜在地用于设计基于机制的抑制剂，有助于减少农业浪费和维持健康的营养。以时间依赖的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，特别是在需要远距离运输农产品或在缺乏足够冷藏的地区储存农产品的情况下。