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UDP-galactose 4-epimerase from <i>Kluyveromyces fragilis: </i>Equilibrium unfolding studies

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Title UDP-galactose 4-epimerase from Kluyveromyces fragilis: Equilibrium unfolding studies
 
Creator Maity, Nilesh Ranjan
Barat, Bhaswati
Bhattacharyya, Debasish
 
Description 433-441
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 I- 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 ΔG 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.
 
Date 2012-12-31T20:07:25Z
2012-12-31T20:07:25Z
1999-12
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://hdl.handle.net/123456789/15474
 
Language en_US
 
Rights CC Attribution-Noncommercial-No Derivative Works 2.5 India
 
Publisher NISCAIR-CSIR, India
 
Source IJBB Vol.36(6) [December 1999]