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Please use this identifier to cite or link to this item: http://hdl.handle.net/2031/4898

Title: Mechanistic studies of enzymatic reactions involved in fatty acid oxidation
Other Titles: Can yu zhi fang suan dai xie de mei de fan ying ji li yan jiu
參與脂肪酸代謝的酶的反應機理研究
Authors: Zeng, Jia (曾嘉)
Department: Dept. of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2006
Publisher: City University of Hong Kong
Subjects: Enzymes
Fatty acids -- Oxidation
Notes: CityU Call Number: TP248.65.E59 Z46 2006
Includes bibliographical references (leaves 150-173)
Thesis (Ph.D.)--City University of Hong Kong, 2006
xxv, 174, [42] leaves : ill. (some col.) ; 30 cm.
Type: Thesis
Abstract: Numerous diseases have been reported in relation to fatty acids, such as cardiovascular disease, cancer, diabetes, rheumatoid arthritis, fibrosarcoma-induced hyperlipidemia, etc. The regulation of fatty acid oxidation has been reported as a potential method treating non-insulin dependent diabetes mellitus (NIDDM) and inhibitors of enzymes involved in the metabolism of fatty acids have been synthesized and studied as potential medicines. Medium-chain acyl-CoA dehydrogenase (MCAD), acyl-CoA oxidase (ACO) and 3-ketoacyl-CoA thiolase (KT) are three key enzymes involved in the β-oxidation of fatty acid. In the present study, we found that both MCAD and ACO have intrinsic isomerase activities, and carried out extensive studies of MCAD, ACO, and KT through site-directed mutagenesis and incubation with various substrate analogs followed with analysis. We cloned the genes of rat acyl-CoA oxidase, 3-ketoacyl-CoA thiolase and medium-chain acyl-CoA dehydrogenase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the C or N-terminal of the genes respectively. The three cloned genes were overexpressed in Escherichia coli and the soluble proteins were purified with a Hitrap chelating metal affinity column in over 90% yield to apparent homogeneity. MCAD and ACO were found to have intrinsic enoyl-CoA isomerase activity, which were confirmed using incubation followed with HPLC analysis. E376 mutants of MCAD were constructed, and it was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. E421 mutants of ACO were also constructed, and it was shown that E421 is the catalytic residue for both oxidase and isomerase activities of the enzyme. MCAD and ACO may function as isomerase in vivo when authentic isomerase is deficient. As we know, this is the first report that MCAD and ACO have intrinsic enoyl-CoA isomerase activity. Four MCAD mutants Y375A, Y375E, Y375R and Y375K were constructed, and it was found that the mutant Y375K showed intrinsic acyl-CoA oxidase activity, which can transfer electrons to molecular oxygen. 2-Octy-4-enoyl-CoA was found to be a mechanism-based irreversible inhibitor of MCAD, and the mechanism of inactivation was different from those previous reported for known MCAD inhibitors. 2-Octe-4-ynoyl-CoA and 2-pente-4-ynoyl-CoA were both found to be mechanism-based inhibitors of ACO. ACO mutants Y232 and Y401 were constructed for studying the importance of the two residues, and it was found that Y232 and Y401 were important in cofactor FAD binding through kinetic and spectrum studies. Four KT mutants H352A, H352E, H352K and H352Y were constructed, and it was found that all mutants have significantly decreased activity, which confirmed H352 is an essential catalytic residue. KT mutant S251 was also constructed, and it was shown that residue S251 plays an important role in substrate binding. 2-Octynoyl-CoA and 2-octy-4-enoyl-CoA were both found to be mechanism-based inhibitors of KT.
Online Catalog Link: http://lib.cityu.edu.hk/record=b2217971
Appears in Collections:BCH - Doctor of Philosophy

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