Refolding and Reconstitution Studies on the Transacetylase−Protein X (E2/X) Subcomplex of the Mammalian Pyruvate Dehydrogenase Complex:  Evidence for Specific Binding of the Dihydrolipoamide Dehydrogenase Component to Sites on Reassembled E2

Abstract

Reconstitution studies have been conducted on the dihydrolipoamide acetyltransferase-protein X core subcomplex of the mammalian pyruvate dehydrogenase complex. GdnHCl-induced dissociation of this core is an ordered cooperative event involving formation of specific lower-Mr intermediates corresponding to dihydrolipoamide acetyltransferase trimers and monomers. Recovery profiles of the dihydrolipoamide acetyltransferase-protein X core, unfolded in 6 M GdnHCl prior to the removal of denaturant by either (a) slow dialysis or (b) rapid dilution, demonstrated rapid initial reappearance of substantial levels of dihydrolipoamide acetyltransferase activity with complete recovery occurring in 4-6 h. Immunological analysis of reconstituted cores revealed reduced levels of protein X (approximately 30-35%) after slow dialysis and the total absence of this component following rapid dilution. The dihydrolipoamide acetyltransferase core, devoid of protein X, was unable to sustain overall complex activity when reconstituted with stoichiometric amounts of its companion pyruvate decarboxylase and dihydrolipoamide deydrogenase components, whereas the protein X-depleted core could sustain 30-35% of control activity. Further reconstitution analyses of overall complex function with these two types of reassembled core structures in the presence of excess dihydrolipoamide dehydrogenase (100-fold) demonstrated significant additional stimulation of pyruvate dehydrogenase complex activity (25-30%) which was dependent on the source of exogenous dihydrolipoamide dehydrogenase. Thus, this constituent enzyme can interact directly with the dihydrolipoamide acetyltransferase oligomer with low affinity in addition to its normal high-affinity binding to the protein X subunit. These results provide definitive in vitro evidence in support of recent clinical observations reporting residual pyruvate dehydrogenase activity (10-20%) in cell lines derived from patients lacking protein X.

Pyruvate dehydrogenase E3 binding protein deficiency

“…Comparable residual activity has been observed in vitro in PDH complexes depleted of E3BP, suggesting that some functional complex can be formed by a direct interaction between E3 and the E2 core (McCartney et al 1997). This residual activity may be the major factor responsible for the prolonged survival that has been a feature of the majority of patients with E3BP deficiency.…”

Organization of the Cores of the Mammalian Pyruvate Dehydrogenase Complex Formed by E2 and E2 Plus the E3-binding Protein and Their Capacities to Bind the E1 and E3 Components

“…In part, this model was favored based on the results with the yeast system, and the fact that the smallest symmetry element in the dodecahedron has 12 pentagonal faces. In contrast to the yeast system, resolved bovine E3BP, which retained the capacity to bind E3 and had a functional lipoyl domain, failed to bind back to assembled E2 under non-chaotropic conditions or after a rapid transition to less chaotropic conditions (8,14,27,32,33) (see "Discussion").…”

Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex

“…The possible role of species differences in these hybrid complexes was not addressed. The use of heterologous reconstitution systems has recently been questioned [36]. For example, the yeast E3 binds to the bovine kidney E2 core, but does not seem to be oriented properly for catalysis ; it has been suggested that the inability of previous studies to demonstrate PDC activity after selective proteolysis of E3BP might have been the result of the use of heterologous pig heart E3 in the reconstitution studies [37].…”