title: Expression and characterization of the catalytic domain of human phenylalanine hydroxylase.
abstract: A truncated version of human phenylalanine hydroxylase which contains the carboxy terminal 336 amino acids was produced in Escherichia coli. It was purified by ammonium sulfate precipitation, Q-Sepharose chromatography, and hydroxyapatite chromatography. The K(m) values of the truncated enzyme for tetrahydropterin substrates are not different from those of the full-length enzyme, nor are the Vmax values. The KM value for phenylalanine is 2-fold lower for the truncate than for the full-length enzyme. The metal content of the enzyme is 0.27 mol Fe per mole enzyme subunit, and it is activated 2.3-fold by addition of ferrous ion to assays; it is not activated by addition of copper. The truncated enzyme shows no lag in activity when an assay is started with phenylalanine, while the full-length enzyme shows a marked lag.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Daubner
authors.forename: P J
authors.initials: PJ
authors.lastname: Hillas
authors.forename: P F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000032:analysis / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005296:Ferrous Compounds / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000032:analysis; D007700:Kinetics; D010649:Phenylalanine / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010661:Phenylketonurias / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
chemical_list: D005296:Ferrous Compounds; D011994:Recombinant Proteins; D001708:Biopterins; D014443:Tyrosine; D010649:Phenylalanine; D003300:Copper; D007501:Iron; D010651:Phenylalanine Hydroxylase; C003402:sapropterin