Ms. Ghatge et al., UNFOLDING AND REFOLDING OF GLUCOSE XYLOSE ISOMERASE FROM STREPTOMYCESSP NCIM-2730/, Enzyme and microbial technology, 16(4), 1994, pp. 323-327
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.