Abstract
In the previous paper we demonstrated that uridine-5'-beta-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 = 0.2 mM) for UDP-glucose-4-epimerase from Escherichia coli. This active site-directed 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 alpha,beta-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.
Highlights
UDP-glucose-4-epimerase (EC 5.1.3.2, called epimerase) catalyzes a freely reversible reaction between UDPglucose and UDP-galactose
In the previous paper we demonstrated that uridine5Ј-diphosphoro--1-(5-sulfonic acid) naphthylamidate (UDPAmNS)1 in aqueous solution behaves as a quenched fluorophore because of its predominantly stacked conformation
UDPAmNS Is a Probe for the Active Site of E. coli Epimerase—We first investigated whether UDPAmNS can be used as a probe for the active site of the E. coli enzyme
Summary
UDP-glucose-4-epimerase (EC 5.1.3.2, called epimerase) catalyzes a freely reversible reaction between UDPglucose and UDP-galactose This enzyme of the galactose metabolic pathway is essential for the biosynthesis of numerous galactoconjugates in all cell types studied so far. This epimerase has emerged as the prototype of a new class of oxidoreductases in which the coenzyme NAD, noncovalently but firmly bound to the apoenzyme, acts as a true cofactor and not as a cosubstrate as in the case of classical dehydrogenases. It is imperative that modification studies be carried out with the E. coli enzyme to specify and confirm the tentative roles of these amino acid residues residing at the active site. The reliability of these results is confirmed from the available x-ray data
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