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|Title: ||Characterization and inactivation studies of enzymes involved in fatty acid oxidation|
|Other Titles: ||Can yu zhi fang suan dai xie de mei de biao zheng he shi huo yan jiu|
|Authors: ||Wu, Long (武龍)|
|Department: ||Department of Biology and Chemistry|
|Degree: ||Doctor of Philosophy|
|Issue Date: ||2008|
|Publisher: ||City University of Hong Kong|
|Subjects: ||Enzymes -- Biotechnology.|
Fatty acids -- Oxidation.
|Notes: ||CityU Call Number: TP248.65.E59 W83 2008|
xxxiii, 286 leaves : ill. (some col.) 30 cm.
Thesis (Ph.D.)--City University of Hong Kong, 2008.
Includes bibliographical references (leaves 267-286)
|Abstract: ||Numerous diseases have been reported in relation to fatty acids, such as
cardiovascular disease, cancer, diabetes, 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. Mitochondrial trifunctional
protein (MTP), 3-ketoacyl-CoA thiolase (KT), and 2-enoyl-CoA hydratase 2 (ECH 2)
are three key enzymes involved in the β-oxidation of fatty acid. Glutaryl-CoA
dehydrogenase (GCD) and isobutyryl-CoA dehydrogenase (IBD) catalyze the
oxidation of branched chain fatty acids from the catabolism of amino acids.
MTP catalyzes the last three steps of the β-oxidation of long-chain fatty acids. The
3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities reside on the
α-subunit, whereas the 3-ketoacyl-CoA thiolase activity is located on the β-subunit.
This enzyme complex is bound to the mitochondrial inner membrane. Both the α and
the β subunit were overexpressed and purified separately with nickel-metal affinity
column to apparent homogeneity. The pMIS3.0E::β plasmid was then transformed
into competent cells containing pMIS3.0E::α plasmid, and the MTP containing both
the α and the β subunit was overexpressed and purified as a protein complex. FPLC
analysis indicates that the MTP contains two α subunits and two β subunits. Kinetic
studies of the α subunit, the β subunit, and the MTP α2β2 protein complex were carried
out. The results show that all three enzymatic activities including enoyl-CoA
hydratase, 3-hydroxyacyl-CoA dehydrogenase, and acyl-CoA thiolase activities, increased when the α and the β subunit form α2β2 complex. The MTP α2β2 complex
prefers longer chain substrate in both binding capacity and catalytic rate.
(Methylenecyclopropyl)formyl-CoA (MCPF-CoA) was found to be a
mechanism-based irreversible inhibitor of the α subunit, while trimetazidine and
oct-2-yn-4-enoyl-CoA were found to be two mechanism-based irreversible inhibitors
of the β subunit. The mechanistic studies of the inactivation of the α and the β subunit
by above three inhibitors were carried out. Glu151, Cys105 and Cys424 were found to
be labeled by MCPF-CoA, trimetazidine and oct-2-yn-4-enoyl-CoA, respectively.
KT catalyzes the last step reaction of the β-oxidation cycle, which involves thiolytic
cleavage of 3-ketoacyl-CoA substrate by free coenzyme A. We found that the enzyme
has intrinsic isomerase activity, which was confirmed using incubation followed with
HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized
through studies of kinetics, substrate specificity, pH dependence, and enzyme
inhibition. Cys382 was identified as the catalytic residue for both thiolase and
isomerase activities of the enzyme. In addition, we found that Cys92 was covalently
labeled by oct-2-ynoyl-CoA. This result clearly demonstrated that oct-2-ynoyl-CoA is
an irreversible inhibitor of the thiolase. This study of selective inactivation of KT by
2-alkynoyl-CoA via its intrinsic isomerase activity provides an example for rationally
developing mechanism-based inhibitors based on a side activity of the enzyme, and
may become a supplemental method for better treatment of cardiovascular disease and
ECH 2 is the middle part of the mammalian peroxisomal multifunctional enzyme
type 2 (MFE-2), which catalyzes the second reaction of the fatty acid β-oxidation.
We cloned the gene of rat ECH 2 to a bacterial expression vector pLM1 with six
continuous histidine codons attached to the N-terminus of the gene. Cloned gene of
ECH 2 was overexpressed in Escherichia coli and purified. MCPF-CoA, oct-3-ynoyl-CoA and oct-2-yn-4-enoyl-CoA were identified as three new irreversible
inhibitors of ECH 2 and Glu47 of ECH 2 was covalently labeled by these inhibitors.
Comparative inhibition studies of ECH 1 and ECH 2 were carried out. This result
indicates ECH1 and ECH2 have certain difference in active site geometry.
Oct-3-ynoyl-CoA may selectively inactivate the β-oxidation in peroxisomes without
significant effect on the β-oxidation in mitochondria.
GCD and IBD are two enzymes involved in oxidation of branched chain fatty acids,
which are in the pathways for the catabolism of lysine and valine, respectively. We
cloned the genes of rat GCD and IBD in a bacterial expression vector pET28a. Cloned
genes of GCD and IBD were overexpressed in Escherichia coli and purified. We
found that oct-4-en-2-ynoyl-CoA and oct-2-ynoyl-CoA are two irreversible inhibitors
of GCD, but these two compounds have no inhibition on IBD. Glu419 was found to be
labeled by oct-4-en-2-ynoyl-CoA and oct-2-ynoyl-CoA. In addition, we also noted that
oct-3-ynoyl-CoA and oct-2-en-4-yn-CoA are two competitive inhibitors of GCD. We
also found that GCD has intrinsic isomerase activity, which was confirmed using
incubation followed with HPLC analysis. IBD did not show this intrinsic isomerase
activity. Glu370 was identified as the catalytic residue for both dehydrogenase and
isomerase activities of the enzyme. Study for straight chain substrate specificity of rat
GCD and IBD was also carried out. The results indicate that the straight chain
substrate pattern of GCD was broader than that of IBD.
Moreover, based on above results, oct-2-yn-4-enoyl-CoA was identified as the first
multifunctional irreversible enzyme inhibitor of fatty acid oxidation, which can
inactivate long-chain fatty acid metabolism in both mitochondria and peroxisomes.|
|Online Catalog Link: ||http://lib.cityu.edu.hk/record=b2339798|
|Appears in Collections:||BCH - Doctor of Philosophy |
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