Stoichiometry, organisation and catalytic function of protein X of the pyruvate dehydrogenase complex from bovine heart.

Mammalian pyruvate dehydrogenase complex (PDC) contains a subunit, protein X, which mediates high-affinity binding of dihydrolipoamide dehydrogenase (E3)to the dihydrolipoamide acetyltransferase (E2) core. Precise stoichiometric determinations on bovine heart PDC, by means of two approaches, indicate the presence of 12 mol protein X/mol PDC and 60 mol E2/mol PDC. Studies of the organisation of collagenase-modified PDC by means of covalent cross-linking of N,N'-1,2-phenylenedimaleimide to lipoamide thiols on protein X, reveal that the main cross-linked products have Mr values corresponding to homodimers of protein X. However, significant formation of higher-Mr aggregates indicates that lipoyl domains of protein X can form an interacting network independent of E2 lipoyl domains. These data suggest that either 12 interacting X monomers or 6 interacting X dimers are involved in the binding of six E3 homodimers to the E2/X core. The presence of 60 E2 subunits/complex also supports proposals for a non-integrated external position of protein X. Collagenase-treated PDC possesses residual activity (15 %), indicating that protein-X-linked lipoamide groups can substitute for the lipoyl domains of E2 in overall complex catalysis. Protein-X-mediated diacetylation of dihydrolipoamide moieties is also performed by the modified complex which raises the possibility of a unique catalytic function for protein X.