Rasmussen U B, Mygind B, Nygaard P
Biochim Biophys Acta. 1986 Apr 11;881(2):268-75. doi: 10.1016/0304-4165(86)90013-9.
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.
通过聚丙烯酰胺凝胶电泳测定，来自大肠杆菌K12的尿嘧啶磷酸核糖基转移酶已被纯化至同质。为此，使用了一个在ColE1质粒上携带upp基因的嘧啶需求菌株，该菌株在粗提物中的尿嘧啶磷酸核糖基转移酶活性高15倍。当该菌株在尿嘧啶饥饿条件下生长时，酶活性又额外提高了10倍。尿嘧啶磷酸核糖基转移酶的分子量测定为75000；该酶由三个分子量为23500的亚基组成。尿嘧啶磷酸核糖基转移酶对尿嘧啶和一些尿嘧啶类似物具有特异性。尿嘧啶和PRib-PP的表观Km值分别为7 microM和300 microM。作为酶活性的效应物，GTP将PRib-PP的Km降低至90 microM，并使Vmax值增加2倍，但对尿嘧啶的Km没有影响。发现GTP的作用是pH依赖性的。尿嘧啶磷酸核糖基转移酶的酶学特性及其合成的调控表明了该酶在嘧啶补救途径中的作用。
