Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12.
Uracil phosphoribosyltransferase from Escherichia coli K12 was purified to homogeneity as determined by polyacrylamide gel electrophoresis. For this purpose a pyrimidine-requiring strain harboring the upp gene on a ColE1 plasmid was used, which showed 15-times higher uracil phosphoribosyltransferase activity in a crude extract. When this strain was grown under conditions of uracil starvation, an additional 10-times elevation of the enzyme activity was obtained. The molecular weight of uracil phosphoribosyltransferase was determined to be 75000; the enzyme consists of three subunits with a molecular weight of 23500. Uracil phosphoribosyltransferase is specific for uracil and some uracil analogues. The apparent Km values for uracil and PRib-PP were 7 microM and 300 microM, respectively. As an effector of enzyme activity, GTP lowered the Km for PRib-PP to 90 microM and increased the Vmax value 2-fold, but had no effect on the Km for uracil. The effect of GTP was found to be pH-dependent. The enzymatic characterization of uracil phosphoribosyltransferase and the observed regulation of its synthesis emphasizes the role of the enzyme in pyrimidine salvage.
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005784:Gene Amplification; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000302:isolation & purification*; D010957:Plasmids; D011743:Pyrimidines / Q000378:metabolism; D013379:Substrate Specificity
D011743:Pyrimidines; D006160:Guanosine Triphosphate; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase