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Dihydrolipoamide dehydrogenase (E3) from Escherichia coli, an FAD-linked homodimer, can be fully reconstituted in vitro following denaturation in 6 M guanidinium chloride. Complete restoration of activity occurs within 1-2 h in the presence of FAD, dithiothreitol, and bovine serum albumin. In the absence of FAD, the dihydrolipoamide dehydrogenase monomer forms a stable folding intermediate, which is incapable of dimerization. This intermediate displays a similar tryptic resistance to the native enzyme but is less heat-stable, because its ability to form native E3 is lost after incubation at 65°C for 15 min. The presence of FAD promotes slow, additional conformational rearrangements of the E3 subunit as observed by cofactor-dependent decreases in intrinsic tryptophan fluorescence. However, after 2 h, the tryptophan fluorescence spectrum and far UV CD spectrum of E3, refolded in the absence of FAD, are similar to that of the native enzyme, and full activity can still be recovered on addition of FAD. Cross-linking studies show that FAD insertion is necessary for the monomeric folding intermediate to attain an assembly competent state leading to dimerization. Thus cofactor insertion represents a key step in the assembly of this enzyme, although its initial presence appears not to be required to promote the correct folding pathway. Dihydrolipoamide dehydrogenase (E3)1 from Escherichia coli is a common component of the pyruvate (PDC) and ␣-ketoglutarate dehydrogenase complexes, which catalyze key steps in carbohydrate metabolism (1, 2). In both cases these multienzyme complexes are composed of non-covalent aggregates of three distinct enzymes, E1, E2, and E3, which act in a concerted fashion to convert their 2-oxoacid substrates to the corresponding acyl-CoAs. The initial oxidative decarboxylation reaction is promoted by a complex-specific thiamine diphosphate -requiring dehydrogenase, which also reductively acylates the lipoamide prosthetic groups covalently-linked to the oligomeric E2 enzyme. Lipoic acid is bound in amide linkage to specific lysine residues located at the tip of exposed type 1 ␤-turns within the flexible N-terminal lipoyl domains of the E2 components, whereas the acyltransferase active sites responsible for transfer of the acyl group to CoA are found near the C termini. In E. coli, as in all organisms studied to date, E3 is a FAD-linked homodimer required for reoxidation of the reduced lipoamide cofactor with NADH as the final reaction product.The genes of the individual enzymes of E. coli PDC were cloned in the early 1980s (3-5), and the complex has been the subject of extensive molecular-genetic, enzymological, and protein engineering studies in the interim period (see Ref. 6 for review). Considerable structural information has been accumulated on the prokaryotic PDCs and ␣-ketoglutarate dehydrogenase complexes in recent years by both elegant multidimensional NMR and x-ray crystallographic techniques (7-13). In the case of the highly conserved dihydrolipoamide dehydrogenase (E3), detailed structur...

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The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M) which cause 40-50% inactivation but no loss of overall secondary or tertiary structures of the individual enzymes; the inactivation of the complex is shown to be caused by dissociation of the E1 and E3 components from the E2/X core assembly. This provides an improved procedure for controlled dissociation of the complex and efficient recovery of its component enzymes in their native states. Higher concentrations of GdnHCl (up to 4 M) lead to the unfolding and irreversible inactivation of the separate enzymes of the complex with the E2/X core proving the most resistant to GdnHCl-induced unfolding. Neither the 60-meric E2/X core assembly nor the dimeric E3 component are dissociated into monomers in the presence of 6 M GdnHCl; the latter enzyme forms higher-M(r) aggregates under these conditions.

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