IN-VITRO RECONSTITUTION OF THE 24-MERIC E2 INNER-CORE OF BOVINE MITOCHONDRIAL BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX - REQUIREMENT FOR CHAPERONINS GROEL AND GROES
Rm. Wynn et al., IN-VITRO RECONSTITUTION OF THE 24-MERIC E2 INNER-CORE OF BOVINE MITOCHONDRIAL BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX - REQUIREMENT FOR CHAPERONINS GROEL AND GROES, Biochemistry, 33(30), 1994, pp. 8962-8968
We have investigated the in vitro reconstitution of the 24-meric inner
core domain (E2c) of the transacylase (E2) component of bovine branch
ed-chain alpha-keto acid dehydrogenase complex. The yield of recombina
nt E2c (amino acid residues 161-421 of bovine E2) expressed in Escheri
chia coli was markedly increased by fusing the bacterial maltose-bindi
ng protein (MBP) to the amino terminus of bovine E2c. Following factor
Xa digestion to remove the MBP moiety, E2e was completely unfolded in
4.5 M guanidine HCl (Gdn.HCl). The denatured E2c monomers (apparent M
(r) = 27 000) were diluted 100-fold at 25 degrees C into a refolding b
uffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins
GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of
the chaperonins in the refolding buffer failed to recover any E2 acti
vity. Recovery of E2 activity obeyed hyperbolic kinetics as a function
of the chaperonin-to-E2c molar ratio and showed a requirement for hyd
rolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in t
he presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissoci
ation of recombinant E2c 24-mers into active trimers was achieved by i
ncubation in 1.5 M Gdn.HCl at 25 degrees C. The E2c trimers with an ap
parent M(r) of 84 000 were isolated by sucrose density gradient centri
fugation in the presence of the chaotropic reagent. Removal of 1.5 M G
dn.HCl resulted in the spontaneous reassembly of trimers into the nati
ve 24-mer structure independent of chaperonins. Our results indicate t
hat in vitro refolding of bovine E2c is a chaperonin-mediated process
and that spontaneous assembly of the 24-meric structure of E2 proceeds
through active trimeric intermediates.