Purification and properties of avian liver p-hydroxyphenylpyruvate hydroxylase.
Avian liver p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) was purified to a 1000-fold increase in specific activity over crude supernatant, utilizing a substrate analogue, o-hydroxyphenylpyruvate, to stabilize the enzyme. The preparation was homogeneous with respect to sedimentation with a sedimentation velocity (s20,w) of 5.3 S. The molecular weight of the enzyme was determined to be 97,000 +/- 5,000 by sedimentation equilibrium, and the molecular weight of the subunits was determined to be 49,000 +/- 3,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis revealed heterogeneity of the purified enzyme. The multiple molecular forms were separable by isoelectric focusing, and their isoelectric points ranged from pH 6.8 to 6.0. The amino acid compositions and tryptic peptide maps of the three forms isolated by isoelectric focusing were very similar. The forms of the enzyme had the same relative activity toward p-hydroxyphenylpyruvate and phenylpyruvate. Conditions which are known to accelerate nonenzymic deamidation of proteins caused interconversion of the multiple molecular forms. Iron was the only transition metal found to be associated with the purified enzyme at significant levels. The amount of enzyme-bound iron present in equilibrium-dialyzed samples was equivalent to 1 atom of iron per enzyme subunit. Purification of the enzyme activity correlated with the purification of the enzyme-bound iron. An EPR scan of the purified enzyme gave a signal at g equal 4.33, which is characteristic of ferric iron in a rhombic ligand field.
D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010105:Oxygenases; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation
D000596:Amino Acids; D007527:Isoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010105:Oxygenases; D015111:4-Hydroxyphenylpyruvate Dioxygenase