1997 Fiscal Year Final Research Report Summary
MOLECULAR MECHANISM OF DIFFERENT ROLES OF MITOCHONDRIAL AND PEROXISOMAL beta-OXIDATION SYSTEMS
| Project/Area Number |
07458160
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
MIURA Retsu Kumamoto Univ.Sch.of Med., Professor, 医学部, 教授 (70093466)
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| Co-Investigator(Kenkyū-buntansha) |
TAMAOKI Haruhiko Kumamoto Univ.Sch.of Med., Instructor, 医学部, 助手 (80264290)
SETOYAMA Chiaki Kumamoto Univ.Sch.of Med., Associate Professor, 医学部, 助教授 (60040250)
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| Project Period (FY) |
1995 – 1997
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| Keywords | acyl-CoA dehydrogenase / acyl-CoA oxidase / beta-oxidation / flavoenzyme / resonance Raman spectroscopy / UV-VIS spectroscopy / D-amino acid oxidase / D-aspartate oxidase |
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| Research Abstract |
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.
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