Reversible folding of UDP-galactose 4-epimerase from Escherichia coli
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
Reversible folding of UDP-galactose 4-epimerase from Escherichia coli
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| Creator |
Dutta, Samrajnee
Maiti, Nilesh Ranjan Bhattacharyya, Debasish |
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| Subject |
Chemistry
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| Description |
UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39-kDa subunits having 1 or 2
molecules of NAD bound non-covalently/dimer. The enzyme can be dissociated and denatured by 8 M
urea at pH 7.0 to a state having only 15% of residual secondary structure. Dilution of the denaturant by
20 mM potassium phosphate, pH 8.5, leads to functional reconstitution of the enzyme. No addition of
extraneous NAD is required for reactivation, indicating a strong affinity of the cofactor for refolded
molecule. The reactivation follows a second-order kinetics ( k = 1.2i0.07XlO' M-' s - ' at 25°C) with
an energy of activation of 23.79 i 0.33 kJ/mol. The native, denatured and renatured states of the enzyme
were characterized by far-ultraviolet CD spectra for secondary structure; protein fluorescence, interaction
with extrinsic fluorescence probe ANS (1 -anilino 8-naphthalene sulfonic acid) and ultraviolet absorption
spectra for tertiary structure and size-exclusion HPLC, gel-filtration chromatography and light-scattering
for quaternary structure. The folding process could be broadly divided into two distinct steps: (a) regain
of secondary structure and dimerization were fast and were complete within 2 min and 9 min, respectively,
and (b) regain of catalytic activity was slow and was complete by 45 min. No active holoenzyme could
be identified. It appears that generation of the NAD-binding site and subsequent assembly of NAD is the
rate-limiting step expressing catalytic activity.
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| Date |
1997
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/736/1/EUROPEAN_JOURNAL_OF_BIOCHEMISTRY%2C_244(_2)%2C_407%2D413[74].pdf
Dutta, Samrajnee and Maiti, Nilesh Ranjan and Bhattacharyya, Debasish (1997) Reversible folding of UDP-galactose 4-epimerase from Escherichia coli. European Journal Of Biochemistry, 244 (2). pp. 407-413. |
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| Relation |
http://dx.doi.org/10.1111/j.1432-1033.1997.t01-1-00407.x
http://www.eprints.iicb.res.in/736/ |
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