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.
Sanderson
S J
SJ
Institute of Biomedical and Life Sciences, University of Glasgow, Scotland.
Miller
C
C
Lindsay
J G
JG
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation; D000818:Animals; D002417:Cattle; D017364:Collagenases / Q000494:pharmacology; D003432:Cross-Linking Reagents; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000502:physiology*; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000502:physiology*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D008063:Thioctic Acid / Q000378:metabolism
D003432:Cross-Linking Reagents; D010455:Peptides; D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; C489061:pyruvate dehydrogenase complex component X, Bos taurus; D009243:NAD; D000105:Acetyl Coenzyme A; D008063:Thioctic Acid; D019289:Pyruvic Acid; D017364:Collagenases