Purification and properties of orotate phosphoribosyltransferases from Escherichia coli K-12, and its derivative purine-sensitive mutant.
Orotate phosphoribosyltransferase (OPT) was purified from both Escherichia coli K-12 strain and its derivative, a purine-sensitive mutant. The wild-type OPT had a molecular weight (M.W.) of 47,000 and was composed of two identical subunits (M.W. 23,500). The wild-type OPT showed maximum activity at pH 9.5, and no activity was seen in the absence of Mg2+ or Mn2+ ion. It also catalyzed a reverse reaction, namely orotidine-5'-monophosphate (OMP) pyrophosphorolysis. In this reverse reaction, tripolyphosphate, tetrapolyphosphate, and trimetaphosphate were also effective as pyrophosphate donors. The apparent Km values of the wild-type OPT were 30 microM for orotate and 40 microM for 5-phosphoribosyl 1-pyrophosphate (PRib-PP), and also 3.6 microM for OMP and 13 microM for PPi. On the other hand, the mutant OPT showed increased apparent Km values for all four substrates, 440 microM for orotate, 360 microM for PRib-PP, 33 microM for OMP, and 250 microM for PPi. The mutant OPT required a higher concentration of Mg2+ ion for maximum activity than the wild-type OPT. The nature of the purine-sensitive phenotype of the mutant is discussed from the standpoint of the reactivity of the mutant OPT, which has an increased Km value for PRib-PP (about 9-fold).
M
M
Shimosaka
Y
Y
Fukuda
K
K
Murata
A
A
Kimura
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D009705:Nucleosides / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D009962:Orotate Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009963:Orotic Acid / Q000378:metabolism; D010430:Pentosyltransferases / Q000302:isolation & purification*; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001426:Bacterial Proteins; D011756:Diphosphates; D009705:Nucleosides; D009711:Nucleotides; D013439:Sulfhydryl Reagents; D008345:Manganese; D009963:Orotic Acid; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D008274:Magnesium