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

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata

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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/