The enzymes of the galactose cluster in Saccharomyces cerevisiae. II. Purification and characterization of uridine diphosphoglucose 4-epimerase.
Uridine diphosphoglucose 4-epimerase (EC 5.1.3.2) of Saccharomyces cerevisiae was purified to homogeneity with a yield of 30%. The purification procedure involved ammonium sulfate precipitation, streptomycin treatment, chromatography on diethylaminoethyl cellulose and hydroxylapatite, and Bio-Gel A-0.5m gel filtration. With the purified enzyme preparation, Km and Vmax values for uridine diphosphogalactose were determined and found to be 0.22 mM and 1.26 mmol/h/mg of protein, respectively. The value of Vmax corresponds to a turnover rate of 3890 molecules of uridine diphosphogalactose converted to uridine diphosphoglucose/min/enzyme molecule. The pH optimum of the enzyme was found to be between 6.8 and 8.0. Amino acid analysis was carried out on the final preparation. Based on the result, the partial specific volume was calculated to be 0.74 ml/g. The NH2-terminal residue of the enzyme was studied by two different methods and found to be threonine. The molecular weight and subunit composition were determined by the combination of the sucrose density gradient centrifugation and gel filtration under nondissociating conditions, and by polyacrylamide gel electrophoresis under dissociating conditions. The results indicated that the enzyme has a molecular weight of 183,000, consisting of two identical subunits. Each molecule of the native enzyme contained 1 molecule of NAD+.
D000596:Amino Acids / Q000032:analysis; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005690:Galactose / Q000378:metabolism*; D007700:Kinetics; D012440:Saccharomyces / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013045:Species Specificity; D014534:UDPglucose 4-Epimerase / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase; D005690:Galactose