Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599175
Title: Structural enzymology of 2-oxo acid dehydrogenases : symmetry and multiplicity
Author: Frank, R. A. W.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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Abstract:
The 2-oxo acid dehydrogenase multienzyme complexes are found in all the taxonomic domains of life and are compared of three different component enzymes (E1, E2,and E3). Multiple copies of E1 and E3 assemble with E2 en masse generating a particle that eclipses the ribosome (509 MDa). The molecular basis and function of multiplicity and symmetry within these complexes was investigated using the E. coli 2-oxoglutarate dehydrogenase (OGDH, EC 1.2.4.2) and B. stearothermophilus pyruvate dehydrogenase (PDH, EC 1.2.4.1) multienzyme complex. The protein-protein interactions and subunit stoichiometry of the E. coli OGDH multienzyme complex were investigated by limited proteolysis and re-assembly of the recombinant enzyme components (E1, EC 1.2.4.2; E2, EC 2.3.1.61; and E3, EC 1.8.1.4) in vitro. Only four E1 homodimers are able to associate with the E2 24-mer, suggesting that E1-E2 assembly is limited by the available space around the E2 core, rather than the number of binding sites. The four E1 may bind as a tetrahedron about E2, which would be consistent with the cubic symmetry of the core. Cleaving 77 residues from the N-termini of an E1 homodimer inhibits binding to the core domain of E2. In contrast, E3 associates to the peripheral subunit binding domain (PSBD) of E2 and the number of E3 homodimers bound to the E2 complex is also limited to around four despite the availability of 24 PSBD in each E2 complex. The low number of peripheral E1 and E3 components bound to E2 marks the OGDH multienzyme complex apart from all other 2-oxo acid dehydrogenase multienzyme complexes that can be saturated with E1 or E3. Furthermore, the E1 and E3 from many other 2-oxo acid dehydrogenase multienzyme complexes compete for binding to E2, whereas in the E coli OGDH multienzyme complex, the association of E1 and E3 with E2 is non-competitive, which is consistent with their mutually exclusive sites of association on E2. Thus homogenous holo-complexes of E1-E2-E3 were assembled in vitro, each containing 4 E1, 4 E3 and 24 E2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.599175  DOI: Not available
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