GroEL/GroES-dependent reconstitution of alpha(2)beta(2), tetramers of human mitochondrial 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 (similar to 4-5 x 10(3) kD a) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa al pha subunits and two 37.8-kDa beta subunits, In the present study, His(6)-t agged E1 alpha(2)beta(2) tetramers (171 kDa) denatured in 8 M urea were com petently reconstituted in vitro at 23 degrees C with an absolute requiremen t for chaperonins GroEL/GroES and Mg-ATP, Unexpectedly, the kinetics for th e recovery of El activity was very slow with a rate constant of 290 M-1 s(- 1). Renaturation of El with a similarly slow kinetics was also achieved usi ng individual GroEL-alpha and GroEL-beta complexes as combined substrates, However, the beta subunit was markedly more prone to misfolding than the al pha in the absence of GroEL. The alpha subunit was released as soluble mono mers 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 read ily rebound to GroEL when the alpha subunit was absent. Analysis of the ass embly state showed that the His(6)-alpha and beta subunits released from co rresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa alpha beta dimeric intermediate, which subsequently d imerized to produce the active alpha(2)beta(2) tetrameter, The purified alp ha beta dimer isolated from Escherichia coil 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 cy cle until the conversion from dimers to tetramers was complete. Our study d escribes an obligatory role of chaperonins in priming the dimeric intermedi ate for subsequent tetrameric assembly, which is a slow step in the reconst itution of E1 alpha(2)beta(2) tetramers.