COVALENT FLAVIN COENZYMES IN FLAVOENZYME CATALYSIS

这项研究的目标是在分子基础上为 某些新陈代谢重要的黄素酶之所以含有 共价的8α取代的黄素辅酶，而其他的含有 未被取代的黄素辅酶，通过 非共价相互作用。这项研究使用的方法将是 三倍，并将重点放在8Alpha-N(3)-和 8α-N(1)-组氨酸黄素及其所含的酶 功能。第一种方法是深入研究 模型和模型的氧化还原电位与pH的关系 酶结合组氨酸黄酮法测定8α-组氨酸 电离是指改变原子的电子亲和力。 1-电子或2-电子氧化还原反应中的异四氧嘧啶环。这个 第二种方法是使用Endor光谱来监测H和N 与黄素半喹酮偶联，目的是确定 所有自旋密度都分布在8α-咪唑取代基上。 模型和酶系统。第三种方法将是一个详细的 催化该酶的两种黄素酶的比较机理研究 反应(胆固醇氧化酶)，但不同之处在于，一个反应包含一个 8α-N(1)-组氨酸-FAD，而另一个包含带有 意图将任何机械性和氧化还原差异与 8α-N(1)-组氨酸取代基的影响。机械学研究 也将延伸到硫胺素氧化酶，这是一种催化一种 4-电子氧化，含有8α-N(1)-组氨酸-FAD。

The goal of this tudy is to provide arationale, on a molecular basis, as to the reason why certain metabolically important flavoenzymes contain a covalent 8alpha-substituted flavin coenzyme while others contain an unsubstituted flavin coenzyme which is bound to the apoenzyme by non-covalent interactions. The approaches used in this study will be three-fold and will focus on the 8alpha-N(3)- and 8alpha-N(1)-histidylflavins and the enzymes that contain these functionalities. The first approach will be an in-depth study of the pH-dependence of oxidation-reduction potentials of both model and enzyme-bound 8alpha-histidylflavins to determine if 8alpha-histidyl ionization is involvedin modifying the electron affinity of the isoalloxaine ring either in 1-electron or 2-electron redox reactions. The second approach is to use ENDOR spectroscopy to monitor both H and N coupling to the flavin semiquinone with the intent of determining whether any spin density is distributed on the 8alpha-imidazole substituent in both model and enzyme systems. The third approach will be a detailed comparative mechansistic study of two flavoenzymes which catalyze the same reaction (cholesterol oxidase) but differ in that one contains an 8alpha-N(1)-histidyl-FAD while the other contains "normal" FAD with the intent of correlating any mechanistic and redox differences to the influence of the 8alpha-N(1)-histidyl substituent. Mechanistic studies will also be extended on thiamine oxidase, a flaboenzyme that catalyzes a 4-electron oxidation and contains 8alpha-N(1)-histidyl-FAD.