Purification and characterization of yeast anthranilate phosphoribosyltransferase.
Anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae has been purified to homogeneity from an overproducing strain. Analytical ultracentrifugation demonstrated that the enzyme is a dimer of Mr = 83,000 +/- 4,000 (S20.w = 4.7 S). Moreover, as shown by active enzyme sedimentation, the enzyme remains dimeric even at low concentrations. The presence of yeast phosphoribosylanthranilate isomerase in the gradient does not lead to complex formation between the two enzymes as might be expected if phosphoribosyl anthranilate, the very labile product of the anthranilate phosphoribosyltransferase, were channelled to phosphoribosylanthranilate isomerase in vivo. The steady-state-kinetic behaviour of the enzyme suggests that catalysis involves a ternary enzyme-substrate complex, with KANTm = 1.6 microM, and KPRib-PPm = 22.4 microM. The enzyme has been used to generate phosphoribosylanthranilate in situ for kinetic studies of phosphoribosylanthranilate isomerase from Escherichia coli: KPRAm = 5 microM, kcat = 40 s-1.
Hommel
U
U
Abteilung Biophysikalische Chemie, Universität Basel, Switzerland.
Lustig
A
A
Kirschner
K
K
D019747:Aldose-Ketose Isomerases; D000877:Anthranilate Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002238:Carbohydrate Epimerases / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010430:Pentosyltransferases / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D010430:Pentosyltransferases; D000877:Anthranilate Phosphoribosyltransferase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase; D008274:Magnesium