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

说明(申请人提供)：具有单核非血红素铁活性中心的氧活化酶参与许多代谢重要的反应，这些反应具有巨大的医学、制药和环境意义，催化涉及氧气的广泛反应(单氧化、氧化等)。本提案旨在获得更多关于这种铁酶激活二氧化氧的机制以及辅因子和底物如何在活性部位相互作用以实现催化活性的信息。研究将集中在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，都是重要的。这一建议的具体目的是：1)利用18O动力学同位素效应和稳态前动力学研究ACCO中铁中心的氧活化方式和活性氧化剂的性质；2)使用含有放射性标记的替代底物来破译底物分解机制。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，以及用停流方法表征在催化周转期间积累的中间体。放射性标记底物类似物将用于酶失活研究，这可能被用于设计基于机制的抑制剂，以帮助减少农业废物和维持健康营养。以依赖于时间的方式控制乙烯合成过程的能力可能有助于减少由于过熟造成的农业废物的主要损失。对人类健康的潜在影响是重大的，特别是在需要长途运输农产品或储存在缺乏足够冷藏的地区的情况下。

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.