title: Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.
abstract: Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
authors.forename: S C
authors.initials: SC
authors.lastname: Daubner
authors.forename: D L
authors.initials: DL
authors.lastname: Lohse
authors.forename: P F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D010766:Phosphorylation; D010957:Plasmids; D051381:Rats; D013053:Spectrophotometry; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014446:Tyrosine 3-Monooxygenase