Pyruvate dehydrogenase multienzyme complex. Characterization of assembly intermediates by sedimentation velocity analysis.
The pyruvate dehydrogenase complex is a large, highly organized assembly of several different catalytic and regulatory component enzymes. The structural core of the complex is the E2-X subcomplex, consisting of 60 dihydrolipoamide transacetylase (E2) subunits arranged in a pentagonal dodecahedron; 6 protein X and 2 pyruvate dehydrogenase kinase molecules are tightly associated with this E2 60-mer. The native E2-X subcomplex exhibits a sedimentation coefficient of 32 S. The effects of several chaotropes (guanidinium chloride, potassium thiocyanide, and urea) on the E2-X subcomplex were assessed. Treatment of the E2-X subcomplex with 4 M guanidinium chloride caused a complete loss of enzymatic activity and the dissociation of the subcomplex into monomeric 1.5-3 S species. Removal of the chaotrope by dialysis for 18 h resulted in complete restoration of E2 enzymatic activity and reassembly of a 32 S subcomplex; this reassembled subcomplex contained less protein X than the native subcomplex. Sedimentation velocity analysis of reassembled E2-X subcomplex demonstrated the presence of an 8 S assembly intermediate; this sedimentation coefficient is characteristic of globular proteins of molecular weights similar to that expected for a trimer of E2. Shorter periods of dialysis also gave rise to the 8 S species; the amount of this intermediate decreased with increasing times of dialysis. The 8 S species associated non-cooperatively to yield additional assembly intermediates exhibiting sedimentation coefficients of 10-32 S.
Behal
R H
RH
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
DeBuysere
M S
MS
Demeler
B
B
Hansen
J C
JC
Olson
M S
MS
D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D066298:In Vitro Techniques; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation
D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase