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 sto pped-flow fluorescence and circular dichroism spectroscopy. The effect s of metal ions on the refolding of the native enzyme and the refoldin g of a monomeric mutant ([A208]GDH) have also been studied. The refold ing process of the wild-type enzyme is at least biphasic; 70% of the r espective 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 no t affect the slowest biphasic refolding rate, which is virtually the s ame for all three versions of the enzyme. The presence of GroEL slows down the first phase of refolding. The reassociation of subunits was e xamined by measuring the regain in catalytic activity and the enhancem ent in the fluorescence emission from NADH on binding to the oligomeri c form of the enzyme, The rate and extent of reassociation is dependen t on enzyme concentration and the extent of reactivation is dependent on the presence of the metal ion. The reassociation process was more e fficient in the presence of NADH particularly for the metal-depleted e nzyme (apo-GDH). The presence of GroEL or GroEL plus ATP leads to a hi gher yield of association and therefore catalytically active enzyme. T he additional presence of Mg-ATP does not affect the extent of reassoc iation, 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.