Untersuchungen zum Reaktionsmechanismus einer pflanzlichen Oxidase, von Monooxygenasen und von Modellverbindungen für Dioxygenasen

本课题的主要研究对象是儿茶酚氧化酶、酪氨酸酶和儿茶酚双加氧酶三种金属酶。本项目的目的是研究这三种酶中每一种可能存在的自由基反应途径。邻苯二酚氧化酶和酪氨酸酶是一类双核铜酶，催化单酚或邻苯二酚氧化成邻醌。本课题组测定了甘薯儿茶酚氧化酶的X射线结构。该结构涉及一个不寻常的共价半胱氨酸-组氨酸键接近活性位点表明自由基反应途径的可能性。这一假设应通过EPR测量脱辅基酶以及其他光谱方法和动力学实验来研究。不同的酪氨酸酶也将被分离和结晶。一个完整的单晶X-射线结构测定也打算为了比较这两个双铜酶。酪氨酸酶的反应途径涉及自由基中间体的建议机制将进行详细研究。邻苯二酚双加氧酶是一种非血红素铁酶，催化邻苯二酚芳香环的氧化裂解。这种酶的假设机制涉及短寿命的烷基过氧自由基。从酶和仿生模型复合物的快速动力学研究中间半醌物种应被捕获的自由基，如NO，以深入了解的机制。

The main objects of research in this proposal are the three metalloenzymes catechol oxidase, tyrosinase, and catechol dioxygenase. The aim of this project is to investigate the possible existence of radical reaction pathways for each of these three enzymes. Catechol oxidase and tyrosinase are related dinuclear copper enzymes which catalyze the oxidation of monophenols or o-diphenols to o-quinones. Our group has determined the X-ray structure of catechol oxidase from sweet potato. The structure which involves an unusual covalent cysteine-histidine bond close to the active site indicates the possibility of a radical reaction pathway. This hypothesis shall be investigated by EPR measurements of the apoenzyme as well as by other spectroscopic methods and kinetic experiments. Different tyrosinases will also be isolated and crystallized. A full single crystal X-ray structure determination is also intended in order to compare these two dicopper enzymes. The proposed mechanism for the reaction pathway of tyrosinase involving radical intermediates will be investigated in detail. Catechol dioxygenase is a non-heme iron enzyme catalyzing the oxidative cleavage of the aromatic rings of catechols. The hypothetical mechanism of this enzyme involves short-lived alkyl-peroxy radicals. From fast kinetic studies on the enzyme and on biomimetic model complexes intermediate semiquinone species shall be trapped by radicals such as NO in order to get insight into the mechanism.