UDP-glucose 4-epimerase from Saccharomyces fragilis. Presence of an essential arginine residue at the substrate-binding site of the enzyme.
UDP-glucose 4-epimerase from Saccharomyces fragilis was inactivated by the arginine-specific reagents phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione following pseudo first order reaction kinetics. The reaction order with respect to phenylglyoxal was 1.8 and that with respect to the other two diones was close to unity. Protection afforded by substrate and competitive inhibitors against inactivation by phenylglyoxal and the reduced interaction of 1-anilinonaphthalene 8-sulfonic acid, a fluorescent probe for the substrate-binding region after phenylglyoxal modification, suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of UMP, a ligand known to interact at the substrate-binding region, showed that only the arginine residue at the active site could be modified by phenylglyoxal. The characteristic coenzyme fluorescence of the yeast enzyme was found to be enhanced three times in phenylglyoxal-inactivated enzyme suggesting the incorporation of the phenyl ring near the pyridine moiety of NAD.
D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D001667:Binding, Competitive; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors*; D002250:Carbon Radioisotopes; D003512:Cyclohexanones / Q000494:pharmacology; D003931:Diacetyl / Q000494:pharmacology; D007700:Kinetics; D010658:Phenylglyoxal / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D012440:Saccharomyces / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors*; D014500:Uracil Nucleotides / Q000494:pharmacology
D002250:Carbon Radioisotopes; D003512:Cyclohexanones; D014500:Uracil Nucleotides; C018031:1,2-cyclohexanedione; D001120:Arginine; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase; D003931:Diacetyl; D010658:Phenylglyoxal