title: Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation.
abstract: Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21-41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (Ndelta15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion Ndelta15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (Ndelta30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (Ndelta41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.
authors.affiliation: Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1083, USA.
authors.forename: G J
authors.initials: GJ
authors.lastname: Yohrling
authors.forename: S M
authors.initials: SM
authors.lastname: Mockus
authors.forename: K E
authors.initials: KE
authors.lastname: Vrana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D020319:Oligodeoxyribonucleotides, Antisense; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D020836:Protein Structure, Quaternary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014365:Tryptophan Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D046911:Macromolecular Substances; D020319:Oligodeoxyribonucleotides, Antisense; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014365:Tryptophan Hydroxylase