Rh. Behal et al., PYRUVATE-DEHYDROGENASE MULTIENZYME COMPLEX - CHARACTERIZATION OF ASSEMBLY INTERMEDIATES BY SEDIMENTATION-VELOCITY ANALYSIS, The Journal of biological chemistry, 269(50), 1994, pp. 31372-31377
The pyruvate dehydrogenase complex is a large, highly organized assemb
ly of several different catalytic and regulatory component enzymes. Th
e structural core of the complex is the E2-X subcomplex, consisting of
60 dihydrolipoamide transacetylase (E2) subunits arranged in a pentag
onal dodecahedron; 6 protein X and 2 pyruvate dehydrogenase kinase mol
ecules are tightly associated with this E2 60-mer. The native E2-X sub
complex exhibits a sedimentation coefficient of 32 S. The effects of s
everal chaotropes (guanidinium chloride, potassium thiocyanide, and ur
ea) on the E2-X subcomplex were assessed. Treatment of the E2-X subcom
plex 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 c
omplete 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 intermedi
ate; this sedimentation coefficient is characteristic of globular prot
eins 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 am
ount of this intermediate decreased with increasing times of dialysis.
The 8 S species associated non-cooperatively to yield additional asse
mbly intermediates exhibiting sedimentation coefficients of 10-32 S.