Refolding studies on 2-oxoacid dehydrogenase multienzyme complexes

The 2-oxoacid dehydrogenase multienzyme complexes are high Mr assemblies occupying key positions in intermediary metabolism. Their function is to catalyse the irreversible oxidative decarboxylation of 2-oxoacids by the coordinated action of multiple copies of three separa+e enzymes termed El (2-oxoacid dehydrogenase), E2 (dihydrolipoamide acyltransferase) and E3 (dihydrolipoamide dehydrogenase). The 2- oxoacid dehydrogenase multienzyme complex family consists of the pyruvate dehydrogenase complex (PDC), 2-oxoglutarate dehydrogenase complex (OGDC) and branched chain 2-oxoacid dehydrogenase complex (BCDC). These complexes have been purified from a wide variety of organisms and in all complexes studied, the E2 component forms a symmetrical structural core to which the El and E3 subunits bind. The El and E2 components perform complex specific reaction steps, while the E3 component is responsible for the reoxidation of the lipoamide group in each of the complexes. In eukaryotes, these nuclear encoded complexes are located in the mitochondrion, loosely associated with the inner face of the inner membrane. The in vivo translocation, folding and assembly of such large structures is an intriguing process which is at present not fully understood. This thesis examines the GdnHCl-induced deactivation and subsequent reactivation on dilution from denaturant of OGDC and PDC from bovine heart and PDC from E. coli. Activity is found to be lost at relatively low GdnHCl concentrations in each complex (below 0.3 M). The E. coli PDC is shown to have spontaneous reactivation after higher concentrations of GdnHCl incubation than the bovine heart complexes. The deactivation and reactivation of the individual complex components has also been studied. The E2 component was found to reactivate after higher concentration GdnHCl incubations than the peripheral subunits. The deactivation and reactivation curves of complex intact and isolated components have been compared. For E. coli E3 this revealed a marked difference, with the intact E3 being considerably more stable than its isolated counterpart. This effect is thought to be due to the hydrophobic association of the E3 dimer with the E2 core, which has been shown to be reactivated after higher GdnHCl incubations. The refolding environment of the E3 component has been studied in more detail in Chapter 5. E3 is a member of flavin-containing pyridine nucleotide-disulphide oxidoreductases. All members of this group studied to date are homodimers containing one flavin adenine dinucleotide (FAD) per subunit and a redox active disulphide. Purified E3 from bovine heart, yeast and E. coli have been unfolded using GdnHCl (as determined by CD spectra) and refolding has been attempted under a variety of conditions. The attempts to refold yeast and bovine E3 from a completely unfolded state have proved unsuccessful. The addition of the molecular chaperones groEL and groES (and ATP) to the refolding buffer of E. coli E3 gave a 15% recovery of activity, compared to no recovery when the protein was diluted into buffer alone. The effects of protein concentration and oxidation state of the refolding buffer have also been investigated. The single most important factor, however, has been found to be inclusion of FAD in the refolding buffer. A 5-fold molar excess of FAD (compared to E3 monomers) in the refolding buffer gave a 22% recovery of activity. It is thought that the FAD is creating a folding nucleation site on the E3 protein which encourages the protein down the correct folding pathway towards the native state