ABSTRACT
The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5.5'-disulfonic acid. At slightly higher urea concentrations (0.75-2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first 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 occurs with the formation of intermediate species in which transient higher aggregates appear to be involved.
Subject(s)
Bacillus megaterium/enzymology , Glucose Dehydrogenases/chemistry , Urea/chemistry , Anilino Naphthalenesulfonates , Chromatography, Gel , Circular Dichroism , Cross-Linking Reagents , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes , Glucose 1-Dehydrogenase , Glucose Dehydrogenases/isolation & purification , Protein Denaturation , Protein Folding , Spectrometry, FluorescenceABSTRACT
N-Acetylglucosamine 6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme from Escherichia coli involved in aminosugar catabolism, has been crystallized by the vapour-diffusion technique using phosphate as precipitant. X-ray diffraction experiments show the crystals to belong to the orthorhombic crystal system, with space group P2(1)2(1)2. The unit-cell parameters are a = 82.09 (2), b = 114.50 (1), c = 80.17 (1) A. The crystals diffract to a maximum resolution of 1.8 A and an initial data set was collected to 2.0 A.
