FLAVOPROTEINS IN FATTY ACID METABOLISM

这一提议涉及两种黄素连接酶的作用机制，这两种酶在线粒体脂肪酸氧化和内质网中二硫键形成中发挥作用。酰基-CoA脱氢酶在脂肪酸氧化的遗传缺陷和细胞毒性脂肪酸的生物活化中具有相当大的代谢重要性。 链长歧视的机制将与中链酰基辅酶A脱氢酶，使用各种酰基辅酶A类似物，并采用蛋白质晶体学和动力学探针的活性位点环境进行研究。 将研究影响酶的“开放”和“封闭”构象之间的平衡的因素，以了解催化碱的pK和黄素辅基的反应性是如何调制的。 将检查一系列细胞毒性4-硫代-酰基-CoA类似物对中链酶的基于机制的灭活。 这些共价修饰反应的目标将使用蛋白质化学和晶体学来确定。将使用相同的方法研究几种4-硫代-酰基-CoA类似物对烯酰-CoA水合酶的简易灭活。 来自于鸡蛋白色的巯基氧化酶是一个新认识的蛋白质家族的最佳成员，其在人成纤维细胞、细胞外基质和精子发生中的生长调节中起作用。 该氧化酶对一系列还原蛋白质底物具有高度活性，并与蛋白质二硫键异构酶合作在胰腺核糖核酸酶中产生正确的二硫键配对。 还原性半反应的机制将使用快速反应和静态方法用各种还原的蛋白质底物来探测。 黄素类似物的研究将允许在氧化酶的活性位点环境的评价，将允许调制FAD和氧化还原活性二硫键部分之间的内部平衡，并应提供敏感的分光光度工具的研究还原蛋白质底物和鸡蛋白色氧化酶之间的复合物。

This proposal addresses the mechanism of action of two flavin- linked enzymes with roles in mitochondrial fatty acid oxidation and in disulfide bond formation in the endoplasmic reticulum. The acyl-CoA dehydrogenases have assumed considerable metabolic importance in genetic deficiencies of fatty acid oxidation and in the bio-activation of cytotoxic fatty acids. The mechanism of chain-length discrimination will be investigated with the medium chain acyl-CoA dehydrogenase, using a variety of acyl-CoA analogues, and employing protein crystallography and kinetic probes of the active site environment. Factors influencing the equilibrium between the "open" and "closed" conformations of the enzyme will be studied to understand how the pK of the catalytic base and the reactivity of the flavin prosthetic group are modulated. The mechanism-based inactivation of the medium chain enzyme by a series of cytotoxic 4-thia-acyl-CoA analogues will be examined. The targets of these covalent modification reactions will be determined using protein chemistry and crystallography. The facile inactivation of enoyl-CoA hydratase by several 4-thia- acyl-CoA analogues will be studied using the same methods. The sulfhydryl oxidase from egg white is the best understood member of a newly-recognized protein family with roles in growth regulation in human fibroblasts, in the extracellular matrix, and in spermatogenesis. The oxidase is highly active with a range of reduced protein substrates, and cooperates with protein disulfide isomerase in the generation of the correct disulfide pairings in pancreatic ribonuclease. The mechanism of the reductive half- reaction will be probed with a variety of reduced protein substrates using rapid reaction and static approaches. Flavin analog studies will allow an evaluation of the active site environment in the oxidase, will permit modulation of the internal equilibrium between FAD and redox-active disulfide moieties, and should provide sensitive spectrophotometric tools for the study of complexes between reduced protein substrates and the egg white oxidase.