Structural and Functional Analysis of Component X from the Bovine Heart Pyruvate Dehydrogenase Complex

Sanderson, Sanya J (1994) Structural and Functional Analysis of Component X from the Bovine Heart Pyruvate Dehydrogenase Complex. PhD thesis, University of Glasgow.

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Abstract

A comprehensive investigation of the optimal conditions for reconstitution of bovine heart pyruvate dehydrogenase complex activity (PDC) from dissociated E2/X core and E1/E3 fractions is presented, and was employed in the elucidation of the disputed involvement of the lipoyl domains of protein X in overall complex catalysis. Selective proteolysis of the lipoyl and linker regions of protein X by Arg C, in isolated E2/X core or intact PDC, resulted in a dramatic reduction in E3 binding potential and hence, complex activity (with the former, following reconstitution with dissociated components, El and E3), when compared to control complex activities. It is proposed that owing to the proximity of the cleavage site to the E3 binding domain of protein X, loss of E3 binding affinity arises from conformational changes in the aforementioned domain and/or, the specific binding site within. Partial recovery of reconstitution was achieved in the presence of excess porcine E3, and increased with increasing protein X degradation suggestive of an additional, non specific and hence, low affinity E3 binding site -- presumably the subunit binding domain of E2. Following removal of the lipoyl and linker regions of either component E2 or X, N-ethyl[2,3-14C]maleimide labelling studies confirmed previous observations of a residual (i.e., approx. 10%) complex activity, indicating the ability of either component to substitute for the lipoyl domains of the other. The physiological implications of this are discussed in relation to the unique diacetylation properties of mammalian PDC. However, as attempts to isolate and sequence the extreme C-terminal portion of protein X following the recent discovery of a dihydrolipoamide acetyltransferase active site-like motif by J.C. Neagle (University of Glasgow, unpublished observations), remained in the initial stages, it is unclear whether deacetylation of the lipoyl domains of protein X services an acetyltransferase active site on E2 or X. Precise stoichiometric determinations employing: comparisons of 14C- acetylation in E2 and X; and, densitometric scan analysis of highly purified protein X, E2 and bovine heart PDC samples resolved by SDS-PAGE and silver stained, indicated the presence of 12mol protein X/mol PDC, and of 60mol E2/mol PDC, respectively. Structural studies involving covalent N,N'-1,2-phenylene-dimaleimide (PDM), crosslinking of the lipoyl domains of protein X in E2-lipoyl domain depleted complex, revealed dimeric organisation. Presented together, this evidence is in close agreement with the 1:1 (E3:E1) binding relationship of bovine heart OGDC, and is strongly suggestive of the involvement of six protein X dimers in the binding of six dimers of E3 to the E2/X core in bovine heart PDC. Furthermore, the presence of 60 E2 subunits supports previous proposals for a non-integrated, external protein X core positioning.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J G Lindsay
Keywords: Biochemistry
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-74985
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 14:45
Last Modified: 27 Sep 2019 14:45
URI: http://theses.gla.ac.uk/id/eprint/74985

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