GroEL/GroES-dependent reconstitution of alpha2 beta2 tetramers of humanmitochondrial branched chain alpha-ketoacid decarboxylase. Obligatory interaction of chaperonins with an alpha beta dimeric intermediate.
The decarboxylase component (E1) of the human mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex (approximately 4-5 x 10(3) kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa alpha subunits and two 37.8-kDa beta subunits. In the present study, His6-tagged E1 alpha2 beta2 tetramers (171 kDa) denatured in 8 M urea were competently reconstituted in vitro at 23 degrees 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-alpha and GroEL-beta complexes as combined substrates. However, the beta subunit was markedly more prone to misfolding than the alpha in the absence of GroEL. The alpha subunit was released as soluble monomers from the GroEL-alpha complex alone in the presence of GroES and Mg-ATP. In contrast, the beta subunit discharged from the GroEL-beta complex readily rebound to GroEL when the alpha subunit was absent. Analysis of the assembly state showed that the His6-alpha and beta subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa alpha beta dimeric intermediate, which subsequently dimerized to produce the active alpha2 beta2 tetrameter. The purified alpha beta dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-alpha beta 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-alpha beta 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 alpha2 beta2 tetramers.
Chuang
J L
JL
Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA. chuang01@utsw.swmed.edu
Wynn
R M
RM
Song
J L
JL
Chuang
D T
DT
D002499:Centrifugation, Density Gradient; D018835:Chaperonin 10 / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism*; D018833:Chaperonins / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D014508:Urea
D018835:Chaperonin 10; D018834:Chaperonin 60; D014508:Urea; D018833:Chaperonins; D011767:Pyruvate Decarboxylase