MECHANISMS INVOLVED IN FLAVIN-LINKED OXYGEN METABOLISM

参与分子氧代谢的三种独特黄素蛋白 在链球菌中已经成为本次研究的目标。长期的 该项目的目标是结合对三聚氰胺催化作用的研究 黄素和非黄素氧化还原中心的特异性探针 黄素辅因子与蛋白质环境之间的相互作用。 关于这一机制有两个主要问题 含FAD的NADH过氧化物酶，催化氢的还原 过氧化氢到水中。一种活性部位蛋白的可能参与 该酶催化的氧化还原反应中的二硫化物 用分光光度还原滴定法和化学方法测定 修改。将使用稳态和停流动力学方法。 评价双电子还原酶的催化作用。 四电子转移的NADH氧化酶代表了第二个例子 共同催化中心内相互作用的氧化还原位置。 静态光谱滴定和原子吸收光谱分析 被雇来识别这些地点。此外，半胱氨酸的相关性 含量和酶活性将与分析相结合 超速离心技术确定外源性硫醇在血管紧张素转换酶中的作用 脱黄酮氧化酶的重组。 含有FAD的甘油-3-磷酸氧化酶的不同寻常之处在于 该底物的氧化通常与NAD+的还原相耦合， 而不是分子氧。关于天然酵素是否 展示了典型黄素蛋白的活性部位特征 氧化酶将通过厌氧光还原和滴定 亚硫酸盐。此外，脱辅酶将与人工重组 FAD类似物作为蛋白质环境与蛋白质相互作用的探针 绑住了弗莱文。 链球菌的产能代谢过程，它缺乏 细胞色素和血红素，在很大程度上被忽视了。独特的黄素蛋白 本研究的目标是代表黄素介导的特殊适应 氧化还原功能，使这些生物受益 呼吸活动。

Three unique flavoproteins involved in the metabolism of molecular oxygen in the streptococci have been targeted for this study. The long-term objective of this project is to combine studies of the catalytic roles of flavin and non-flavin oxidation-reduction centers with probes of specific interactions between the flavin cofactor and the protein environment. There are two major questions concerning the mechanism of the FAD-containing NADH peroxidase, which catalyzes the reduction of hydrogen peroxide to water. The possible participation of an active-site protein disulfide in the oxidation-reduction reaction catalyzed by this enzyme will be tested by spectrophotometric reductive titration methods and by chemical modification. Steady-state and stopped-flow kinetic methods will be used to evaluate the catalytic role of the two-electron reduced enzyme. The four-electron transferring NADH oxidase represents a second example of interacting oxidation-reduction sites within a common catalytic center. Static spectral titration methods and atomic absorption spectroscopy will be employed to identify these sites. In addition, correlations of cysteine content and enzyme activity will be combined with analytical ultracentrifugation techniques to define the role of exogenous thiols in the reconstitution of the deflavo oxidase. The FAD-containing glycerol-3-phosphate oxidase is unusual in that the oxidation of this substrate is generally coupled to the reduction of NAD+, not of molecular oxygen. The question of whether the native enzyme exhibits the active-site characteristics of the typical flavoprotein oxidases will be tested by anaerobic photoreductions and by titrations with sulfite. In addition, the apoenzyme will be reconstituted with artificial FAD analogs as probes of the interaction between protein environment and bound flavin. The energy-yielding metabolic processes of the streptococci, which lack cytochromes and hemes, have been largely ignored. The unique flavoproteins targeted in this study represent special adaptations of flavin-mediated oxidation-reduction function which allow these organisms a beneficial respiratory activity.