Fluorescence properties of reconstituted forms of UDP-glucose 4-epimerase from Saccharomyces fragilis.
UDP-glucose 4-epimerase from Saccharomyces fragilis exhibits a very characteristic intense fluorescence with an excitation maximum at 360 nm and an emission maximum at 433 nm. The fluorescence spectrum resembles the fluorescence of free NADH with an apparent blue shift and, although the exact nature of the fluorophore is not known, the protein-bound NAD, which is a coenzyme for this reaction, or its reduced form is obviously involved in the emission of the fluorescence. The fluorphore therefore constitutes part of the active site. The inactivation of epimerase with diazinedicarboxylic acid bis(N,N-diethylamide), a reaction shown in the previous paper to form a disulfide linkage across the subunits, results in a simultaneous and correlated loss of the characteristic fluorescence of the enzyme. Reaction with mercaptoethanol restores the native fluorescence with a 2 nm blue shift in emission maximum. These epxeriments provide additional evidence that the two conformationally vicinal sulfhydryl groups are located at the active site. Unlike the reconstituted enzyme obtained from the diamide-inactivated enzyme, the partialy active enzymes reconstituted from p-chloromercuribenzoate-inactivated and heat-inactivated enzymes fail to show the reappearance of the characteristic native fluorescence. Treatment with N-ethylmaleamide, on the other hand, leads to a form of the inactive enzyme that fully retains its fluorescent properties. A model depicting the minimal changes at the active site during the process of inactivation and reconstitution by these various treatments is presented.
D002238:Carbohydrate Epimerases / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D011489:Protein Denaturation; D012440:Saccharomyces / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014534:UDPglucose 4-Epimerase / Q000378:metabolism*
D003958:Diamide; D008623:Mercaptoethanol; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase