UDP-galactose 4-epimerase from Kluyveromyces fragilis : Reconstitution of Holoenzyme Structure After Dissociation With Parachloromercuribenzoate
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
UDP-galactose 4-epimerase from Kluyveromyces fragilis : Reconstitution of Holoenzyme Structure After Dissociation With Parachloromercuribenzoate
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| Creator |
Majumdar, Siddhartha
Bhattacharjee, Hiranmoy Bhattacharyya, Debasish Bhaduri, Amar Nath |
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| Subject |
Chemistry
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| Description |
UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis (Kluyveromyces marxianus var. marxianus)
is a homodimer of molecular mass 75 kDa/subunit and has one mol NAD firmly bound/dimer. The
pathway for the assembly of the holoenzyme structure has been studied after dissociating the native
epimerase with p-chloromercuribenzoate into inactive mercurated monomers. The process of dissociation
was not associated with unfolding of the molecules. Reconstitution of the functional holoenzyme was
done by reduction with dithiothreitol and addition of extra NAD. The reaction was thus followed to
monitor maturation of the enzyme from the folded monomeric state. The reconstituted enzyme was similar
to the native enzyme in terms of a number of physiochemical properties such as secondary, tertiary
and quarternary structures, Km for the substrate UDP-galactose, reductive inhibition, interaction with the
fluorophore 1-anilino 8-naphthalene sulphonic acid (ANS), etc. Reconstitution under low ionic strength
buffer (I 5 0.011) shows that the presence of NAD is essential for the formation of a dimeric structure.
However, dimeric apoenzyme could also be stabilized under high ionic strength buffer (I 5 0.1). Reactivation
was strongly dependent on pH, being most effective at pH 8.1. Kinetic evidence suggested that, at
low ionic strength, assembly of NAD over dimeric apoenzyme is the rate-limiting step in expressing
catalytic activity. This process has a low energy of activation of 27.2 kJ/mol.
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| Date |
1998
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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/854/1/EUROPEAN_JOURNAL_OF_BIOCHEMISTRY%2C_257(_2)%2C_427%2D433_[17].pdf
Majumdar, Siddhartha and Bhattacharjee, Hiranmoy and Bhattacharyya, Debasish and Bhaduri, Amar Nath (1998) UDP-galactose 4-epimerase from Kluyveromyces fragilis : Reconstitution of Holoenzyme Structure After Dissociation With Parachloromercuribenzoate. European Journal Of Biochemistry, 257 (2). pp. 427-433. |
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| Relation |
http://dx.doi.org/10.1046/j.1432-1327.1998.2570427.x
http://www.eprints.iicb.res.in/854/ |
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