FLAVOPROTEINS IN FATTY ACID METABOLISM

这项建议阐述了两种黄素连接酶的作用机制，它们在线粒体脂肪酸氧化和内质网二硫键形成中发挥作用。酰辅酶A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激活中具有相当重要的代谢作用。利用中链酰辅酶A脱氢酶，使用各种酰基辅酶A类似物，并利用蛋白质结晶学和活性中心环境的动力学探针来研究链长区分的机制。影响酶“开放”和“封闭”构象之间平衡的因素将被研究，以了解催化碱基的PK和黄素修复基团的反应活性是如何被调节的。我们将研究一系列细胞毒性4-硫酰-辅酶A类似物对中链酶失活的机理。这些共价修饰反应的目标将用蛋白质化学和结晶学来确定。我们将用同样的方法研究几种4-硫酰辅酶A类似物对Enoyl-CoA水合酶的简易失活。蛋清中的巯基氧化酶是一个新发现的蛋白质家族中最被理解的成员，它在人成纤维细胞、细胞外基质和精子发生中发挥着生长调节的作用。该酶与一系列还原的蛋白质底物有很高的活性，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对。使用快速反应和静态方法，将用各种还原的蛋白质底物来探索还原半反应的机理。黄素类似物的研究将允许评估酶中的活性中心环境，将允许调节FAD和氧化还原活性二硫键之间的内部平衡，并将为研究还原蛋白底物和蛋清氧化酶之间的络合物提供灵敏的分光光度工具。

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.