An arginine residue is essential for stretching and binding of the substrate on UDP-glucose-4-epimerase from Escherichia coli - Use of a stacked and quenched uridine nucleotide fluorophore as probe

In the previous paper we demonstrated that uridine-5'-beta-1-(5-sulfonic ac id) naphthylamidate (UDPAmNS) is a stacked and quenched fluorophore that sh ows severalfold enhancement of fluorescence in a stretched conformation. UD PAnNS was found to be a powerful competitive inhibitor (K-i = 0.2 mM) for U DP-glucose-4-epimerase from Escherichia coli, This active site-directed flu orophore assumed a stretched conformation on the enzyme surface, as was evi denced by full enhancement of fluorescence in saturating enzyme concentrati on. Complete displacement of the fluorophore by UDP suggested it to bind to the substrate binding site of the active site. Analysis of inactivation ki netics in presence of alpha,beta-diones such as phenylglyoxal, cyclohaxaned ione, and a,3-butadione suggested involvement of the essential arginine res idue in the overall catalytic process. From spectral analysis, loss of acti vity could also be directly correlated with modification of only one argini ne residue. Protection experiments with UDP showed the arginine residue to be located in the uridyl phosphate binding subsite, Unlike the native enzym e, 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.