GroEL, GroES, and ATP-dependent folding and spontaneous assembly of ornithine transcarbamylase.
When purified rat liver ornithine transcarbamylase (OTC), a trimer of 36 kDa subunits, was denatured in 6 M guanidine hydrochloride and then diluted 50-100-fold, no activity was recovered, and the OTC subunits aggregated. In contrast, when the chaperonin groEL was included in the dilution buffer, OTC did not aggregate but instead comigrated in a sucrose density gradient with the groEL oligomer, indicating that a complex had been formed. Upon addition of the cochaperonin groES and ATP to the isolated OTC-groEL complex, OTC monomers were folded, released, and assembled into active trimer. Neither groES nor ATP alone was sufficient to release active OTC from groEL. The extent of recovery of activity was proportional to the concentration of the complex, reaching approximately 80-90% at monomer concentrations above 0.6 microM. At low complex concentrations, kinetic studies revealed an initial lag in the reconstitution reaction, suggesting that assembly is the rate-limiting step under these conditions. We could trap folded, released, inactive OTC monomers at early times that assembled into active trimers with longer incubation. A nonhydrolyzable ATP analog could release bound OTC from groEL in the presence of groES, but the OTC monomers were not competent for assembly. These data show that recovery of OTC activity in vitro can be efficiently directed by the bacterial chaperonins in the presence of ATP and suggest that the mechanism of reconstitution involves ATP and groES-dependent folding and release of OTC monomers from groEL, followed by spontaneous assembly of trimers.
Zheng
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X
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Rosenberg
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LE
Kalousek
F
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Fenton
W A
WA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D051381:Rats
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; D009954:Ornithine Carbamoyltransferase