Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation

The effect of urea on glucose dehydrogenase from Bacillus megaterium has be en studied by following changes in enzymatic activity, conformation and sta te of aggregation. It was found that the denaturation process involves seve ral transitions. At very low urea concentrations (below 0.5 M), where the e nzyme is fully active and tetrameric, there is a conformational change as m onitored by an increase in intensity of the tryptophan fluorescence and a m aximum exposure of organized hydrophobic surfaces as reported by the fluore scence of 4,4'-dianilino-1,1'-binaphthyl-5.5'-disulfonic acid. At slightly higher urea concentrations (0.75-2 M), a major conformational transition oc curs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the fo rmation of interacting intermediates that lead to a highly aggregated state . Increasing urea concentrations cause a complete dissociation to lead firs t a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase o ccurs with the formation of intermediate species in which transient higher aggregates appear to be involved. (C) 2000 Elsevier Science B.V. All rights reserved.