Expression and characterization of the catalytic domain of human phenylalanine hydroxylase

S. Colette Daubner, Patrick J. Hillas, Paul F. Fitzpatrick

Research output: Contribution to journalArticle

32 Scopus citations

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 V(max) values. The K(M) 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.

Original languageEnglish (US)
Pages (from-to)295-302
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume348
Issue number2
DOIs
StatePublished - Dec 15 1997

Keywords

  • Domains
  • Kinetics
  • Mutagenesis
  • Phenylalanine hydroxylase
  • Phenylketonuria
  • Tetrahydrobiopterin

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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