Expression and characterization of the catalytic domain of human phenylalanine hydroxylase

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 S-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. (C) 1997 Academic Press.