title: UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies.
abstract: UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.
authors.affiliation: Division of Protein Engineering, Indian Institute of Chemical Biology, Jadavpur, Calcutta.
authors.forename: N R
authors.initials: NR
authors.lastname: Maity
authors.forename: B
authors.initials: B
authors.lastname: Barat
authors.forename: D
authors.initials: D
authors.lastname: Bhattacharyya
mesh_terms: D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D007716:Kluyveromyces / Q000201:enzymology*; D011489:Protein Denaturation; D013816:Thermodynamics; D014534:UDPglucose 4-Epimerase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D014534:UDPglucose 4-Epimerase