GroEL/GroES-dependent reconstitution of α2β2 tetramers of human mitochondrial branched chain α-ketoacid decarboxylase: Obligatory interaction of chaperonins with an αβ dimeric intermediate

Jacinta L. Chuang, R. Max Wynn, Jiu Li Song, David T. Chuang

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28 Scopus citations


The decarboxylase component (E1) of the human mitochondrial branched chain α-ketoacid dehydrogenase multienzyme complex (~4-5 x 103 kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa α subunits and two 37.8-kDa β subunits. In the present study, His6-tagged E1 α2β2 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-α and GroEL-β complexes as combined substrates. However, the β subunit was markedly more prone to misfolding than the α in the absence of GroEL. The α subunit was released as soluble monomers from the GroEL-α complex alone in the presence of GroES and Mg-ATP. In contrast, the β subunit discharged from the GroEL-β complex readily rebound to GroEL when the α subunit was absent. Analysis of the assembly state showed that the His6-α and β subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa αβ dimeric intermediate, which subsequently dimerized to produce the active α2β2 tetrameter. The purified αβ dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-αβ 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-αβ 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 α2β2 tetramers.

Original languageEnglish (US)
Pages (from-to)10395-10404
Number of pages10
JournalJournal of Biological Chemistry
Issue number15
StatePublished - Apr 9 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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