Multiple unfolded states of UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis. Involvement of proline cis-trans isomerization in reactivation.
UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis is a dimeric molecule of 75 kDa per subunit with one molecule of cofactor NAD per dimer. It undergoes unfolding and complete dissociation in presence of 8 M urea at pH 7.0 by 10 min. It can be functionally reconstituted almost quantitatively in 2 h by dilution with 20 mM sodium phosphate buffer, pH 7 containing 1 mM extraneous NAD under a second order kinetics [Bhattacharyya, D. (1993) Biochemistry 32, 9726-9734]. Denaturation between 10-60 min inversely affects both the rate and maximum recovery of activity upon refolding. Aggregation of this protein has not been observed under these conditions. The time dependent reaction at the unfolded state is independent of pH between 5.4-10.4 but strongly dependent on temperature of denaturation between 0-20 degrees C. Unfolding at 0 degrees C divides the protein largely into two populations-34% of fast folding species following an apparent first order kinetics and 59% of slow folding species following a second order kinetics of reactivation. A very fast folding species of low abundance 3.5-7.5% depending on temperature of denaturation has been identified, which gets active status within the dead time of mixing. Interaction with the active site directed fluorescence probe 1-anilino 8-naphthalene sulfonic acid (1-ANS) and estimation of bound NAD suggest that the catalytic region of this enzyme is not formed in the long term denatured samples. The whole process of reactivation is catalysed by peptidyl prolyl cis-trans isomerase and thus suggests that one or more proline residues stereochemically control the rate limiting step of reactivation.
Dutta
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S
Indian Institute of Chemical Biology, Jadavpore, Calcutta.
Maity
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NR
Bhattacharyya
D
D
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002850:Chromatography, Gel; D019281:Dimerization; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014534:UDPglucose 4-Epimerase / Q000737:chemistry* / Q000378:metabolism; D014508:Urea
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D009243:NAD; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D011392:Proline; D014534:UDPglucose 4-Epimerase; D019696:Peptidylprolyl Isomerase