COVALENT FLAVIN COENZYMES IN FLAVOENZYME CATALYSIS

本研究的目的是在分子基础上， 某些代谢上重要黄素酶含有 共价的8 α-取代的黄素辅酶，而其他的含有 未取代的黄素辅酶，其通过以下方式与脱辅基酶结合： 非共价相互作用。 本研究中使用的方法将是 三倍，并将重点放在8 α-N（3）-和 8 α-N（1）-组氨酰黄素和含有这些黄素的酶 功能。 第一种方法将是深入研究 pH依赖性的氧化还原电位的模型和 酶结合的8 α-组氨酰黄素，以确定8 α-组氨酰 电离涉及到改变电子亲合势， 异咯烷环在1-电子或2-电子氧化还原反应中。 的 第二种方法是使用ENDOR光谱来监测H和N 与黄素半醌偶联，目的是确定是否 任何自旋密度都分布在8 α-咪唑取代基上， 模型和酶系统。 第三种方法将是详细的 两种黄素酶催化相同酶的比较机理研究 反应（胆固醇氧化酶），但不同的是，其中一个包含一个 8 α-N（1）-组氨酰-FAD，而另一种含有“正常”FAD， 将任何机制和氧化还原差异与 8 α-N（1）-组氨酰取代基的影响。 机制研究 也将延伸到硫胺素氧化酶，一种催化 4-电子氧化并含有8 α-N（1）-组氨酰-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.