UNFOLDING AND REFOLDING OF GLUCOSE XYLOSE ISOMERASE FROM STREPTOMYCESSP NCIM-2730/

The unfolding and refolding of the tetrameric D-glucose/xylose isomera se (GXI) from Streptomyces sp. NCIM 2730 has been investigated by corr elating the biological activity with the protein transitions as monito red by fluorometry, c.d., and by its retention volumes in molecular si eve chromatography. Treatment of the enzyme with SDS (0.1%) resulted i n the dissociation of the tetramer (T) into an active dimer (D) with n o gross change in the tertiary structure but with change in the second ary structure. On removal of the denaturant, a pars' of the dimer reas sociated to form a small amount of tetramer. Incubation of the enzyme with Gdn. HCl (2M) resulted in the formation of an inactive dimer (D) that had conformational properties of a molten globule, namely, a nat ive-like secondary structure and disordered tertiary structure. Regain ing of activity was observed on lowering of the concentration of the d enaturant by dilution. Refolding of the Gdn-HCl-treated enzyme resulte d in the restoration of its tertiary structure and activity. The enzym e was completely inactivated by heating at 100 degrees C for 5 min. Th e heated enzyme is a monomer and exhibits a distinct irreversible chan ge in its structure. Thus, four distinct species have been identified and characterized during denaturation and renaturation of the GXI: (i) native tetramer (T), (ii) active and inactive dimers (D & D), and (i ii) inactive monomer (M). The results suggest that the intact tertiary rather than the secondary structure is essential for GXI activity.