The isolation and properties of phenylalanine hydroxylase from rat liver.
Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.
D000818:Animals; D001667:Binding, Competitive; D002374:Catalase; D002614:Chelating Agents; D002850:Chromatography, Gel; D003300:Copper; D004229:Dithiothreitol; D004589:Electrophoresis, Disc; D005182:Flavin-Adenine Dinucleotide; D005461:Fluorine; D007501:Iron; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D008982:Molybdenum; D010544:Peroxidases; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase / Q000302:isolation & purification*; D011622:Pterins; D051381:Rats; D013438:Sulfhydryl Compounds; D014461:Ultracentrifugation
D002614:Chelating Agents; D011622:Pterins; D013438:Sulfhydryl Compounds; D005182:Flavin-Adenine Dinucleotide; D005461:Fluorine; D010649:Phenylalanine; D003300:Copper; D008982:Molybdenum; D007501:Iron; D010544:Peroxidases; D002374:Catalase; D010651:Phenylalanine Hydroxylase; D004229:Dithiothreitol