UDP-galactose 4-epimerase from Kluyveromyces fragilis. Evidence for independent mutarotation site.
UDP-galactose 4-epimerases from the yeast Kluyvero-myces fragilis and Escherichia coli are both homodimers but the molecular mass of the former (75 kDa/subunit) is nearly double that of the latter (39 kDa/subunit). Protein databank sequence homology revealed the possibility of mutarotase activity in the excess mass of the yeast enzyme. This was confirmed by three independent assay protocols. With the help of specific inhibitors and chemical modification reagents, the catalytic sites of epimerase and mutarotase were shown to be distinct and independent. Partial proteolysis with trypsin in the presence of specific inhibitors, 5'-UMP for epimerase and galactose for mutarotase, protected the respective activities. Similar digestion with double inhibitors cleaved the molecule into two fragments of 45 and 30 kDa. After separation by size-exclusion HPLC, they manifested exclusively epimerase and mutarotase activities, respectively. Epimerases from Kluyveromyces lactis var lactis, Pachysolen tannophilus and Schizosaccharomyces pombi also showed associated mutarotase activity distinct from the constitutively formed mutarotase activity. Thus, the bifunctionality of homodimeric yeast epimerases of 65-75 kDa/subunit appears to be universal. In addition to the inducible bifunctional epimerase/mutarotase, K. fragilis contained a smaller constitutive monomeric mutarotase of approximately 35 kDa.
Brahma
Amrita
A
Division of Drug Development, Design and Molecular Modeling, Indian Institute of Chemical Biology, Calcutta, India.
Bhattacharyya
Debasish
D
D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D017510:Protein Folding; D013379:Substrate Specificity; D014534:UDPglucose 4-Epimerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase; C019646:aldose 1-epimerase