Open AccessStoichiometry, Organisation and Catalytic Function of Protein X of the Pyruvate Dehydrogenase Complex from Bovine Heart
Author(s) -
Sanderson Sanya J.,
Miller Clare,
Lindsay J. Gordon
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00068.x
Subject(s) - stoichiometry , pyruvate dehydrogenase complex , catalysis , chemistry , function (biology) , biochemistry , enzyme , biology , microbiology and biotechnology , organic chemistry
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 cros‐linking of N,N′ ‐1,2‐phenylenedimaleimide to lipoamide thiols on protein X, reveal that the main cross‐linked products have M r values corresponding to homodimers of protein X. However, significant formation of higher‐ M r 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.