Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299965
Title: Protein-protein interactions within the 2-oxoacid dehydrogenase complexes
Author: Richards, Susan Diane
ISNI:       0000 0001 3517 1422
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1999
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Abstract:
The 2-oxoacid dehydrogenase complexes; pyruvate dehydrogenase (PDC), 2- oxoglutarate dehydrogenase (OGDC) and branch-chain 2-oxoacid dehydrogenase (BCOADC), are mitochondrial multienzyme complexes which comprise of multiple copies of three different enzymes; El, E2 and E3, that catalyse the oxidative decarboxylation of 2-oxoacids to their respective acyl CoA derivatives. The E2 enzyme forms the structural core, to which several copies of both El and E3 are non-covalently bound. Despite being three of the largest known proteins, relatively little is known regarding the interactions between the constituent components. This is due primarily to the flexibility within the complex which makes the generation of large quantities of material difficult. In this thesis, we have demonstrated the ability to overexpress various subgenes of the complexes in E. coli. The first is a sub-gene of the E2-PDC enzyme from various sources and also the corresponding sub-gene of protein X, termed the di-domain. These di-domains, are comprised of the peripheral subunit binding domain and adjacent lipoyl domain. We have shown that the di-domains have retained their ability to fold correctly, by studying their lipoylation state. Their function to bind the E3 enzyme has been demonstrated by surface plasmon resonance. By binding purified E3 enzymes to the surface of a dextran-coated chip and passing crude extracts containing the overexpressed di-domains over the E3, we have determined that the strongest binding affinities were between human E3 and the protein X di-domain, further substantiating the hypothesis that protein X has evolved to bind E3. The binding affinities between E2-PDC and E3 are sufficiently high to suggest that in the absence of competition from El, E3 may be able to form a stable complex with E2. The sequences surrounding the lipoylation site, a conserved DKA motif within the lipoyl domain, have been investigated using a monoclonal Ab specific for the lipoylated form of the domain, to determine residues critical for recognition by the lipoylating enzymes. To date, there has been some ambiguity with regard to the requirements of the lipoylating enzymes to enable lipoylation to occur. We have shown, with the use of lipoyl domain point mutants that the levels of lipoylation can be altered by mutating residues close to the critical lipoyl lysine. The structure of OGDC has evolved with significant structural differences to PDC and BCOADC. In particular, the El component has the ability to interact directly with E3 as well as the E2 core. The N-terminal region of El is thought to be important in these interactions. Subsequently we have overexpressed, in E. coli, three N-terminal fragments for use as competitive inhibitors of OGDC reconstitution. Despite failing to bind E3, these fragments display inhibition following MgCl2 dissociation, suggesting interference with El binding to the E2 core. Future work is planned to further investigate the divergent structure and protein-protein interactions of OGDC, along with interactions within the other two complexes. The overexpression and subsequent purification of each individual component should allow progress in this area.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.299965  DOI: Not available
Keywords: Multienzyme complexes
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