PMID:11989664
| Citation |
Nayar, S and Bhattacharyya, D (2001) UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies. Indian J. Biochem. Biophys. 38:353-60 |
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| Abstract |
UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation. Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here. These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition. The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure. Thermodynamical parameters associated with some transitions have been quantitated. The results have been discussed with the X-ray crystallographic structure of the enzyme. |
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| Keywords |
Binding Sites; Chromatography, High Pressure Liquid; Circular Dichroism; Dimerization; Enzyme Reactivators/pharmacology; Escherichia coli/enzymology; Kinetics; NAD/metabolism; NAD/pharmacology; Protein Conformation; Protein Denaturation; Protein Folding; Thermodynamics; UDPglucose 4-Epimerase/antagonists & inhibitors; UDPglucose 4-Epimerase/chemistry; UDPglucose 4-Epimerase/isolation & purification; UDPglucose 4-Epimerase/metabolism |
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