GroEL/GroES-dependent Reconstitution of α2β2 Tetramers of Human Mitochondrial Branched Chain α-Ketoacid Decarboxylase

Abstract

The decarboxylase component (E1) of the human mitochondrial branched chain -ketoacid dehydrogenase multienzyme complex (4 -5 10 3 kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa subunits and two 37.8-kDa subunits. In the present study, His 6 -tagged E1 2 2 tetramers (171 kDa) denatured in 8 M urea were competently reconstituted in vitro at 23 C with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP. Unexpectedly, the kinetics for the recovery of E1 activity was very slow with a rate constant of 290 M 1 s 1 . Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL- and GroEL- complexes as combined substrates. However, the subunit was markedly more prone to misfolding than the in the absence of GroEL. The subunit was released as soluble monomers from the GroEL- complex alone in the presence of GroES and Mg-ATP. In contrast, the subunit discharged from the GroEL- complex readily rebound to GroEL when the subunit was absent. Analysis of the assembly state showed that the His 6 - and subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa dimeric intermediate, which subsequently dimerized to produce the active 2 2 tetrameter. The purified dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL- ternary complex. Incubation of this novel ternary complex with GroES and Mg-ATP resulted in recovery of E1 activity, which also followed slow kinetics with a rate constant of 138 M 1 s 1 . Dimers were regenerated from the GroEL- complex, but they needed to interact with GroEL/GroES again, thereby perpetuating the cycle until the conversion from dimers to tetramers was complete. Our study describes an obligatory role of chaperonins in priming the dimeric intermediate for subsequent tetrameric assembly, which is a slow step in the reconstitution of E1 2 2 tetramers.