Presence of two conformationally vicinal sulfhydryl groups at the active site of UDP-glucose 4-epimerase from Saccharomyces fragilis.
UDP-glucose 4-epimerase from Saccharomyces fragilis was inactivated by diazene dicarboxylic acid bis-N,N-dimethylamide or diamide, a compound that can specifically oxidize conformationally vicinal sulfhydryl groups on protein surfaces. The inactive enzyme was shown to retain the original dimeric structure and NAD, which is a coenzyme for this reaction, was not dissociated from the apoenzyme. The loss of activity was due to the direct modification of sulfhydryl groups and could not be attributed to any subsequent loss of structural integrity. The activity of the enzyme could be regained almost completely on incubation with mercaptoethanol alone and no exogenous NAD was needed for reactivation. The reactivated enzyme showed most of the characteristic properties of the native enzyme like activation by cations or inhibition by UMP. Presence of substrate provided partial protection against inactivation by the reagent. Formation of disulfide bond(s) across the subunits was demonstrated by sodium dodecyl sulfate gel electrophoresis in absence of mercaptoethanol. Titration of native and diamide-inactivated enzyme with p-chloromercuribenzoate revealed that only two sulfhydryl groups were involved in the formation of the disulfide cross-linkage across the subunits. The above results indicate the possible presence of two conformationally vicinal sulfhydryl groups at two different subunits of the enzyme that constitute part of the active site.
D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D012440:Saccharomyces / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis; D014534:UDPglucose 4-Epimerase / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D003958:Diamide; D008623:Mercaptoethanol; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase