<span style="font-size:14.0pt;font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-fareast-language: EN-US;mso-bidi-language:AR-SA">UDP-galactose 4-epimerase from <i>Escherichia coli: </i>Equilibrium unfolding studies</span>

Abstract

UDP-galactose 4-epimerase from <i>Escherichia coli </i>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 <i>M </i>urea at <i style="mso-bidi-font-style:normal">p</i>H 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 f1uorophore I-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 <i>M </i>urea; an active monomeric

holoenzymc at 3 <i>M </i>urea with only 40% of secondary structure and finally further denaturation by 6 <i>M </i>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.