Denaturation of the Bacillus stearothermophilus Dihydrolipoamide Dehydrogenase in the Presence of Guanidine-HCl at Low Temperature

Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase induced by incubation with guanidine-HCl (GdnHCl) at 4℃ was examined. Enzyme activity was amplified by 2.5 times in 0.2M GdnHCl; such an increase in activity was also detected in NaCl and KCl at concentrations less than 1M, but not in urea. With increasing amount of GdnHCl, the enzyme lost half and all its activity in 1.0M and 1.6M GdnHCl, respectively. Notable changes in fluorescence specta of Trp residue and FAD cofactor to the inactivation. Based on changes in fluerescence intensities and molecular ellipicity, a two-state transition equilibrium was suggested; transition midpoints were roughly at 1.7M GdnHCl at concentrations above 2M, the enzyme released FAD and formed inactive aggragate. Effects by GdnHCl at concentrations less than 1.4M were mostly chancelled by its removal. Most results were similar to those from studies on the enzyme being components of pyruvate dehdrogenase complex

Effects of Guanidine Hydrochloride on Pyruvate Dehydrogenase Complex

Many molecules of pyruvate decarboxylase （E1） and dihydrolipoamide dehydrogenase （E3） bind non-covalently to a sixty-meric lipoate acetyltransferase （E2）, forming the Bacillus stearothermophilus pyruvate dehydrogenase complex. Changes in the complex induced by incubation in guanidine hydrochloride （GdnHCl） solution and those induced by the incubation followed by removal of GdnHCl were examined. Small amount of GdnHCl induced reversible changes in the complex. The removal re-associate all components to the complex and restoned the enzyme activities of the complex and its components. NaCl induced similar changes, and the effect of GdnHCl was implied as a salt rather than a denaturant. On the other hand, larger amount of GdnHCl induced irreversible changes in the complex. E1, E2, and E3 were dissociated and inactivated; FAD was released from E3. GdnHCl at concentrations limited to a small range around 1.0 M induced a partial and irreversible aggregation. The removal of GdnCHl yielded an E2-E3 complex （57S), restoring most activities of E2 and E3. Loss in enzyme activity of E1 might be responsible for that of the complex