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

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、药学和环境意义的重要代谢反应，催化涉及分子氧的广泛反应（单加氧、氧化等）。目前的建议旨在获得更多的信息，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，使催化活性。研究将集中于1-氨基环丙烷-1-羧酸氧化酶（ACCO），一种独特的抗坏血酸依赖性非血红素铁酶，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）使用18 O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; 2）通过使用含有放射性标记的替代底物来解释底物分解机制。一系列的研究计划，包括18 O动力学同位素效应的测量，以确定还原剂结合的顺序和抗坏血酸在反应中的作用，和表征的中间体，积累在催化周转停流方法。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以有助于减少由于过熟而造成的农业废物的主要损失。对人类健康的潜在影响很大，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农产品的情况下。

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.