Subunit and catalytic component stoichiometries of an in vitro reconstituted human pyruvate dehydrogenase complex.
The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772-19780).
Brautigam
Chad A
CA
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. chad.brautigam@utsouthwestern.edu
Wynn
R Max
RM
Chuang
Jacinta L
JL
Chuang
David T
DT
D001665:Binding Sites; D002384:Catalysis; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D051047:Dihydrolipoyllysine-Residue Acetyltransferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000737:chemistry* / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014461:Ultracentrifugation
D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D030481:Pyruvate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase; D051047:Dihydrolipoyllysine-Residue Acetyltransferase