FLAVOPROTEINS IN FATTY ACID METABOLISM

点击翻译按钮获取中文摘要

DESCRIPTION: This proposal focuses mainly on the acyl-CoA dehydrogenases and metabolically related enzymes. These flavoproteins have assumed considerable metabolic importance in genetic deficiencies of fatty acid oxidation in humans and in the bioactivation of cytotoxic fatty acids. Three fundamental aspects of acyl-CoA dehydrogenase catalysis will be addressed. First, the role of hydrogen bonding in the polarization of the carbonyl group of acyl-CoA thioesters will be probed using FAD and substrate analogs. These studies will also address the importance of H-bonds in the stabilization of enolate species. Second, the role of desolvation, protonation state and electrostatic interactions within the active site of the acyl-CoA dehydrogenase will be examined by static and rapid reaction studies using native and mutant proteins. The modulation of the oxygen reactivity of flavoproteins is very poorly understood, and so a third goal is to use site directed and random mutagenesis studies to assess whether the acyl-CoA dehydrogenase can be converted to an oxidase by increasing solvent accessibility to the FAD cofactor. Correspondingly, the role of TRP166 in the interaction between the dehydrogenase and electron transfer flavoprotein will be probed. A fourth aim addresses the mode of action of cytotoxic 4-thia-fatty acid derivatives of halogenated hydrocarbons such as trichloroethylene. The corresponding CoA thioesters are activated by the acyl-CoA dehydrogenase with the release of highly reactive alpha-halo-thiolate species. Characterization of the reactivity of these compounds is important in understanding the cytotoxicity and mutagenicity of these species. Finally, the ultimate oxidant for disulfide bond formation in secreted proteins is still unclear. A new flavoenzyme oxidase isolated from egg white will be characterized and its potential role in disulfide generation will be studied.