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

本课题的主要研究对象是三种金属酶儿茶酚氧化酶、酪氨酸酶和儿茶酚双加氧酶。本项目的目的是研究这三种酶的自由基反应途径的可能存在。儿茶酚氧化酶和酪氨酸酶是相关的双核铜酶，它们催化单酚或邻二酚氧化为邻醌。本课题组测定了甘薯中儿茶酚氧化酶的x射线结构。该结构涉及一个不寻常的共价半胱氨酸-组氨酸键靠近活性位点，表明了自由基反应途径的可能性。这一假设将通过对脱酶的EPR测量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迪铜酶。本文将对酪氨酸酶涉及自由基中间体的反应途径的机制进行详细的研究。儿茶酚双加氧酶是一种催化儿茶酚芳香环氧化裂解的非血红素铁酶。这种酶的假设机制涉及短寿命的烷基过氧自由基。通过对酶和仿生模型配合物的快速动力学研究，研究中间半醌类物质被NO等自由基捕获的机理。

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.