Refolding and reassociation of glycerol dehydrogenase from Bacillus stearothermophilus in the absence and presence of GroEL.
The refolding of the tetrameric, metalloenzyme glycerol dehydrogenase (GDH) from Bacillus stearothermophilus has been investigated using stopped-flow fluorescence and circular dichroism spectroscopy. The effects of metal ions on the refolding of the native enzyme and the refolding of a monomeric mutant ([A208]GDH) have also been studied. The refolding process of the wild-type enzyme is at least biphasic; 70% of the respective signal changes occur in the first 2 ms followed by a slower process with a half-life of 3 s. The presence of the metal ion does not affect the slowest biphasic refolding rate, which is virtually the same for all three versions of the enzyme. The presence of GroEL slows down the first phase of refolding. The reassociation of subunits was examined by measuring the regain in catalytic activity and the enhancement in the fluorescence emission from NADH on binding to the oligomeric form of the enzyme. The rate and extent of reassociation is dependent on enzyme concentration and the extent of reactivation is dependent on the presence of the metal ion. The reassociation process was more efficient in the presence of NADH particularly for the metal-depleted enzyme (apo-GDH). The presence of GroEL or GroEL plus ATP leads to a higher yield of reassociation and therefore catalytically active enzyme. The additional presence of Mg-ATP does not affect the extent of reassociation, but has a small positive effect on the rate of reassociation. These data suggest that GDH is bound weakly to GroEL and that GroES is not required for release of the protein.
Department of Biochemistry, University of Southampton, England.
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Gore
D000255:Adenosine Triphosphate / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism*; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018834:Chaperonin 60; D000255:Adenosine Triphosphate; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase