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An Arginine Residue Is Essential for Stretching and Binding of the Substrate on UDP-glucose-4-epimerase from Escherichia coli

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Title An Arginine Residue Is Essential for Stretching and Binding of the
Substrate on UDP-glucose-4-epimerase from Escherichia coli
 
Creator Bhattacharyya, Uma
Dhar, Gautam
Bhaduri, Amar Nath
 
Subject Infectious Diseases and Immunology
 
Description In the previous paper we demonstrated that uridine-
5*-b-1-(5-sulfonic acid) naphthylamidate (UDPAmNS) is
a stacked and quenched fluorophore that shows severalfold
enhancement of fluorescence in a stretched conformation.
UDPAmNS was found to be a powerful competitive
inhibitor (Ki 5 0.2 mM) for UDP-glucose-4-
epimerase from Escherichia coli. This active sitedirected
fluorophore assumed a stretched conformation
on the enzyme surface, as was evidenced by full enhancement
of fluorescence in saturating enzyme concentration.
Complete displacement of the fluorophore
by UDP suggested it to bind to the substrate binding site
of the active site. Analysis of inactivation kinetics in
presence of a,b-diones such as phenylglyoxal, cyclohaxanedione,
and 2,3-butadione suggested involvement of
the essential arginine residue in the overall catalytic
process. From spectral analysis, loss of activity could
also be directly correlated with modification of only one
arginine residue. Protection experiments with UDP
showed the arginine residue to be located in the uridyl
phosphate binding subsite. Unlike the native enzyme,
the modified enzyme failed to show any enhancement of
fluorescence with UDPAmNS clearly demonstrating the
role of the essential arginine residue in stretching and
binding of the substrate. The potential usefulness of
such stacked and quenched nucleotide fluorophores has
been discussed.
 
Publisher American Society for Biochemistry and Molecular Biology
 
Date 1999
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/682/1/JOURNAL_OF_BIOLOGICAL_CHEMISTRY__274_(21_14573%2D14578;1999[51].pdf
Bhattacharyya, Uma and Dhar, Gautam and Bhaduri, Amar Nath (1999) An Arginine Residue Is Essential for Stretching and Binding of the Substrate on UDP-glucose-4-epimerase from Escherichia coli. The Journal of Biological Chemistry, 274 (21). pp. 14573-14578.
 
Relation http://dx.doi.org/10.1074/jbc.274.21.14573
http://www.eprints.iicb.res.in/682/