COVALENT FLAVIN COENZYMES IN FLAVOENZYME CATALYSIS

该项目的目标是寻求对分子的理解， 一些重要的代谢物质 氧化还原酶需要8 α-取代的， 共价结合的黄素辅酶，而其它的没有。 的 所使用的方法包括氧化还原的比较研究， 这两种胆固醇氧化酶的催化特性， 有一个正常的FAD，而其他包含8 α-N（1）- 组氨酰FAD 氧化还原电位与 将对胆固醇氧化酶、硫胺素氧化酶和 8 α-取代的FMN-脱辅基黄素氧还蛋白复合物， 延伸到线粒体琥珀酸脱氢酶， 8 α-N（3）-组氨酰基FAD。 机制研究利用 稳态和停流动力学技术将在 肝单胺氧化酶;一种神经药理学酶 生物胺调节的重要性，其中含有8 α-S-半胱氨酸FAD。 圆二色性和ENDOR 光谱方法将用于监测 蛋白质构象对黄素还原的影响，并探讨 在几种不同的环境中， 酶系统 8 α-取代基在一个 电子转移反应将采用停流动力学方法进行探测 重构脱辅基黄素氧还蛋白-8 α FMN复合物的研究 细胞色素C。 这些研究的结果将是 在线粒体电子传递和 阐明重要酶的作用机制 系统如琥珀酸脱氢酶和单胺氧化酶。

The goal of this project is to seek an understanding on a molecular basis as to the reason why certain metabolically important oxidation-reducton enzymes require an 8 alpha-substituted, covalently bound flavin coenzyme while others do not. The approaches to be used include a comparative study of redox and catalytic properties of the two cholesterol oxidases, one which has a normal FAD while the others contains an 8 alpha-N(1)- histidyl FAD. The dependence of oxidation-reduction potential on pH will be done on the cholesterol oxidases, thiamine oxidase and 8 alpha-substituted FMN-apoflavodoxin complexes and will be extended to mitochondrial succinate dehydorgenasewhich contains an 8 alpha-N(3)-histidyl FAD. Mechanistic studies utilizing steady-state and stopped-flow kinetic techniques will be done on liver monoamine oxidase; an enzyme of neuro-pharmacological importance in biogenic amine regulation which contains an 8 alpha-S-cysteinly FAD. Circular dichroism and ENDOR spectroscopic approaches will be used to monitor alterations in protein conformation on flavin reduction and probe the environments of the respective covalent flavin moieties in several enzyme systems. The role of the 8 alpha-substituent in one- electron transfer reactions will be probed by stopped-flow kinetic studies on reconstituted apoflavodoxin-8 alpha FMN complexes with cytchrome c. The results of these studies will be of importance in mitochondrial electron transport and in the elucidation of the mechanism of action of important enzyme systems such as succinate dehydrogenase and monoamine oxidase.