EVIDENCE FOR RESIDUAL STRUCTURES IN AN UNFOLDED FORM OF YEAST PHOSPHOGLYCERATE KINASE

The unfolding-refolding transition of phosphoglycerate kinase followed by steady-state fluorescence has clearly shown the existence of a hyp erfluorescent form [Missiakas et al. (1990) Biochemistry 29, 8683-8689 ]. In order to determine the contribution of each of the two tryptopha ns to the fluorescence properties of the enzyme in the equilibrium tra nsition and to characterize the hyperfluorescent form, two single tryp tophan mutants in which tryptophans 308 and 333 were replaced by a tyr osine and a phenylalanine, respectively, were constructed. Neither the catalytic nor the physicochemical properties of the enzyme are signif icantly altered by these mutations. The unfolding-refolding transition s were studied using circular dichroism and tryptophan fluorescence em ission. Both tryptophans contribute to the hyperfluorescence observed in the first transition. For guanidine hydrochloride concentrations hi gher than 0.9 M, it clearly appears that the second transition results from a further unfolding. It is accompanied by a decrease in fluoresc ence intensity and a 5 nm red shift of the maximum emission wavelength . When the unfolding is induced by urea, the end of the transition cor responds to the hyperfluorescent state. Further addition of guanidine hydrochloride induces complete unfolding. These results suggest the pr esence of residual microstructures around tryptophan 308 and tryptopha n 333 in the hyperfluorescent state. The characterization of these clu sters and their contribution as starting structures in the folding pro cess are now under investigation.