Characterization of chimeric pterin-dependent hydroxylases: contributions of the regulatory domains of tyrosine and phenylalanine hydroxylase to substrate specificity.
Tyrosine and phenylalanine hydroxylases contain homologous catalytic domains and dissimilar regulatory domains. To determine the effects of the regulatory domains upon the substrate specificities, truncated and chimeric mutants of tyrosine and phenylalanine hydroxylase were constructed: Delta117PAH, the C-terminal 336 amino acid residues of phenylalanine hydroxylase; Delta155TYH, the C-terminal 343 amino acid residues of tyrosine hydroxylase; and 2 chimeric proteins, 1 containing the C-terminal 331 residues of phenylalanine hydroxylase and the N-terminal 168 residues of tyrosine hydroxylase, and a second containing the C-terminal 330 residues of tyrosine hydroxylase and the 122 N-terminal residues of phenylalanine hydroxylase. Steady-state kinetic parameters with tyrosine and phenylalanine as substrate and the need for pretreatment with phenylalanine for full activity were determined. The truncated proteins showed low binding specificity for either amino acid. Attachment of either regulatory domain greatly increased the specificity, but the specificity was determined by the catalytic domain in the chimeric proteins. All three proteins containing the catalytic domain of phenylalanine hydroxylase were unable to hydroxylate tyrosine. Only wild-type phenylalanine hydroxylase required pretreatment with phenylalanine for full activity with tetrahydrobiopterin as substrate.
Daubner
S C
SC
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Hillas
P J
PJ
Fitzpatrick
P F
PF
D000595:Amino Acid Sequence; D000818:Animals; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D010651:Phenylalanine Hydroxylase; D014446:Tyrosine 3-Monooxygenase