UDP-Galactose 4-Epimerase from KluyVeromyces fragilis: Analysis of Its Hysteretic Behavior during Catalysis
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
UDP-Galactose 4-Epimerase from KluyVeromyces fragilis: Analysis of Its
Hysteretic Behavior during Catalysis
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| Creator |
Nayar, Suprabha
Brahma, Amrita Barat, Bhaswati Bhattacharyya, Debasish |
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| Subject |
Structural Biology & Bioinformatics
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| Description |
UDP-galactose 4-epimerase serves as a prototype model of class II oxidoreductases that use
bound NAD as a cofactor. This enzyme from KluyVeromyces fragilis is a homodimer with a molecular
mass of 75 kDa/subunit. Continuous monitoring of the conversion of UDP-galactose (UDP-gal) to UDPglucose
(UDP-glu) by the epimerase in the presence of the coupling enzyme UDP-glucose dehydrogenase
and NAD shows a kinetic lag of up to 80 s before a steady state is reached. The disappearance of the lag
follows first-order kinetics (k ) 3.22 � 10-2 s-1) at 25 °C at enzyme and substrate concentrations of
1.0 nM and 1 mM, respectively. The observed lag is not due to factors such as insufficient activity of the
coupling enzyme, association or dissociation or incomplete recruitment of NAD by epimerase, product
activation, etc., but was a true expression of the activity of the prepared enzyme. Dissociation of the
bound ligand(s) by heat followed by analysis with reverse-phase HPLC, TLC, UV-absorption spectrometry,
mass spectrometry, and NMR showed that in addition to 1.78 mol of NAD/dimer, the epimerase also
contains 0.77 mol of 5¢-UMP/dimer. The latter is a strong competitive inhibitor. Preincubation of the
epimerase with the substrate UDP-gal or UDP-glu replaces the inhibitor and also abolishes the lag, which
reappeared after the enzyme was treated with 5¢-UMP. The lag was not observed as long as the cells were
in the growing phase and galactose in the growth medium was limiting, suggesting that association with
5¢-UMP is a late log-phase phenomenon. The stoichiometry and conserved amino acid sequence around
the NAD binding site of multimeric class I (classical dehydrogenases) and class II oxidoreductases, as
reported in the literature, have been compared. It shows that each subunit is independently capable of
being associated with one molecule of NAD, suggestive of two NAD binding sites of epimerase per
dimer.
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| Date |
2004
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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/501/1/BIOCHEMISTRY%2C_43(_31)%2C__10212%2D10223__[37].pdf
Nayar, Suprabha and Brahma, Amrita and Barat, Bhaswati and Bhattacharyya, Debasish (2004) UDP-Galactose 4-Epimerase from KluyVeromyces fragilis: Analysis of Its Hysteretic Behavior during Catalysis. Biochemistry , 43 (31). pp. 10212-10223. |
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| Relation |
http://dx.doi.org/10.1021/bi049569t
http://www.eprints.iicb.res.in/501/ |
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