超氧阴离子自由基从黄素蛋白NrdI传递到半乳糖氧化酶（GAO）促进GAO酶活提高的机理研究

半乳糖氧化酶(GAO)在生物传感器、制备药物中间体和化学品有广泛应用。在GAO催化过程中，伴随着活性中心辅因子被氧化与被还原，辅因子经历由“Cu(2+)-酪氨酸自由基”配位体到“Cu(1+)-酪氨酸”配位体的循环转换，GAO展现出“有活性-无活性-有活性”的循环转换。促进（激活）该循环转换将极大地提高半乳糖氧化酶的催化活性。申请人研究组前期实验研究结果表明黄素蛋白NrdI与GAO共同催化能够极大地提高GAO活性。NrdI能够把溶解氧还原为超氧阴离子自由基。基于前期实验结果以及GAO和NrdI的特点，本项目提出超氧阴离子自由基从NrdI传递到GAO活性中心并氧化“Cu(1+)-酪氨酸”配位体为“Cu(2+)-酪氨酸自由基”配位体的研究构思，拟研究超氧阴离子自由基传递并促进GAO酶活提高机理，并研究多酶协同催化机制。期望通过实施本项目，所构建的多酶体系具工业应用潜力。

Galactose oxidase (GAO) has a wide applications in biopharmaceuticals, biosensors, and preparation of sugar derivatives and chemicals. During GAO catalysis, the cofactor of copper-TyrO radical experiences from being reduced to being oxidized, conferring the enzyme a cycle of active-inactive-active. Promoting the cycle can significantly enhance the activity of GAO. We have carried out experiments demonstrating that Flavoprotein NrdI can enhance the activity of GAO significantly. NrdI can reduce O2 to superoxide anion radical. Based the experimental work and the characteristic of GAO and NrdI, this project suggests the transfer of superoxide anion radical from NrdI to the active site of GAO, and oxidize “Cu(1+)-Tyr” to “Cu(2+)-TyrO”. This project will study the mechanism of superoxide anion radical transfer and the promoting the GAO activity. Furthermore, the coordination of multienzyme will be studied. The results of the project will provide basic research results that can be learned when studying other enzymes. Hopefully, the established multienzyme systems can have a potential of industrial application.