MOLECULAR MECHANISM OF DIFFERENT ROLES OF MITOCHONDRIAL AND PEROXISOMAL beta-OXIDATION SYSTEMS

脂肪酸在体内通过b-氧化途径被氧化，b-氧化途径发生在线粒体或过氧化物体中。这些途径的起始和限速步骤是由线粒体acy1-CoA脱氢酶(ACD)和过氧化物体acy1-CoA氧化酶(ACO)催化的，这两种酶都是FAD依赖的黄素酶。本课题利用紫外可见光谱、共振拉曼光谱和核磁共振光谱研究了底物类似物与ACD和ACO的相互作用模式。所使用的底物类似物是具有不同酰基1链长度的3-酮酰基-辅酶A，并且已知与ACD和ACO形成电荷转移络合物，在可见光谱中具有独特的宽吸收带特征。详细分析了可见光光谱中最大吸收峰的形状、强度和波长，并测量和分析了电荷转移络合物在电荷转移带内的共振喇曼光谱。这些酶的底物专一性反映在底物类似物与结合黄素的电荷转移相互作用上。配体与ACD和ACO的相互作用方式在两种酶之间略有不同，但差异很大，说明酶与底物相互作用的方式存在细微的差异。根据在特定位置富含C-13同位素的配体的核磁共振谱推测了底物活化机理。从活性中心结构和底物与黄素的相互作用两个方面解释了ACD和ACO之间的细微差别。我们还对D-氨基酸氧化酶和D-天冬氨酸氧化酶进行了研究。这些酶和ACO一样定位于过氧化物体中。这些酶的晶体分析、光谱测量和动力学分析为了解其活性中心结构、底物结合方式和底物活化机理提供了重要信息。

Fatty acids are oxidized in vivo by b-oxidation pathways, which occur in either mitochondria or peroxisomes. The initial and rate-limiting steps of these pathways are catalyzed by mitochondrial acy1-CoA dehydrogenase (ACD) and peroxisomal acy1-CoA oxidase (ACO), both of which are FAD-dependent flavoenzymes. In the present project, the interaction modes of substrate analogs with ACD and ACO were analyzed by means of UV-VIS,resonance Raman and NMR spectroscopy. The substrate analogs used are 3-ketoacyl-CoA's with various acy1-chain lengths and are known to form charge-transfer complexes with ACD and ACO which are characterized by unique broad absorption band in the long wave-length region in VIS spectra. The shapes, intensity, wavelength of the maximum absorption in VIS spectra are carefully analyzed and resonance Raman spectra of the charge-transfer complexes with excitation within the charge-transfer band were measured and analyzed. The substrate specificity of these enzymes are reflected in the charge-transfer interactions of the substrate analogs with the bound flavin. The interacting modes of the ligands in with ACD and ACO are slightly but distinctly different between the two enzymes, indicating the subtle difference in the enzyme-substrate interactions. The substrate-activating mechanism was deduced from the NMR spectra of the ligand enriched with C-13 isotopes at specific positions. The subtle differences observed between ACD and ACO are interpreted in terms of the active-site structure and substrate-flavin interaction.We have also undertaken investigation on D-amino acid oxidase and D-aspartate oxidase. These oxidases are localized in peroxisomes as is ACO.Crystallographic analysis, spectroscopic measurement and kinetical analysis of these oxidases have provided important information on the active-site structure, mode of substrate binding and substrate-activating mechanism.