Abstract

The urea-induced unfolding at pH 7.5 of Escherichia coli phosphoenolpyruvate (P-pyruvate) carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism spectra, and 1-anilino-8-naphthalenesulfonate binding. These studies were performed in the absence and presence of substrates and ligands. ATP or P-pyruvate plus MnCl2, or of the combined presence of ATP plus MnCl2 and oxalate, conferred great protection against urea-induced denaturation. The unfolding process showed the presence of at least one stable intermediate which is notably shifted to higher urea concentrations in the presence of substrates. This intermediate protein structure was inactive, contained less tertiary structure than the native protein and retained most of the original secondary structure. Hydrophobic surfaces were more exposed in the intermediate than in the native or unfolded species. Refolding experiments indicated that the secondary structure was completely recovered. Total recovery of tertiary structure and activity was obtained only from samples denatured at urea concentrations lower than these where the intermediate accumulates.