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.