Carboxyl terminal deletion analysis of tryptophan hydroxylase

Susan M. Mockus, Sean C. Kumer, Kent Vrana

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the synthesis of serotonin and participates (in a non-rate-limiting fashion) in melatonin biosynthesis. In rabbit, TPH exists as a tetramer of four identical 51,007 dalton (444 amino acids) protein subunits. An intersubunit binding domain responsible for tetramer formation of TPH was identified by assessing the role of a carboxyl terminal leucine heptad and 4-3 hydrophobic repeat. These repeats are conserved in all of the aromatic amino acid hydroxylases and have been shown to be required for the assembly of tyrosine hydroxylase tetramers. Polymerase chain reaction was utilized to create three TPH carboxyl terminal deletions (C Δ8, C Δ12 and C Δ17) that sequentially remove members of the leucine heptad and 4-3 hydrophobic repeat. Each deletion and full-length recombinant;TPH was expressed in bacteria to obtain soluble enzyme extracts for subsequent activity and structural analysis. It was found that removal of 8, 12 or 17 amino acids from the carboxyl terminus of TPH did not significantly alter enzymatic activity when compared to full-length recombinant TPH. However, the macromolecular structure of the deletions was dramatically affected as determined by dimeric and monomeric profiles on size exclusion chromatography. It can be concluded that amino acids 428-444 (the C-terminal 17 amino acids) comprise an intersubunit binding domain that is required for tetramer formation of TPH, but that tetramer assembly is not essential for full enzymatic activity.

Original languageEnglish (US)
Pages (from-to)132-140
Number of pages9
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1342
Issue number2
DOIs
StatePublished - Oct 17 1997

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Tryptophan Hydroxylase
Amino Acids
Leucine
Aromatic Amino Acids
Size exclusion chromatography
Polymerase chain reaction
Biosynthesis
Protein Subunits
Tyrosine 3-Monooxygenase
Melatonin
Mixed Function Oxygenases
Structural analysis
Gel Chromatography
Serotonin
Bacteria
Rabbits
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

Cite this

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title = "Carboxyl terminal deletion analysis of tryptophan hydroxylase",
abstract = "Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the synthesis of serotonin and participates (in a non-rate-limiting fashion) in melatonin biosynthesis. In rabbit, TPH exists as a tetramer of four identical 51,007 dalton (444 amino acids) protein subunits. An intersubunit binding domain responsible for tetramer formation of TPH was identified by assessing the role of a carboxyl terminal leucine heptad and 4-3 hydrophobic repeat. These repeats are conserved in all of the aromatic amino acid hydroxylases and have been shown to be required for the assembly of tyrosine hydroxylase tetramers. Polymerase chain reaction was utilized to create three TPH carboxyl terminal deletions (C Δ8, C Δ12 and C Δ17) that sequentially remove members of the leucine heptad and 4-3 hydrophobic repeat. Each deletion and full-length recombinant;TPH was expressed in bacteria to obtain soluble enzyme extracts for subsequent activity and structural analysis. It was found that removal of 8, 12 or 17 amino acids from the carboxyl terminus of TPH did not significantly alter enzymatic activity when compared to full-length recombinant TPH. However, the macromolecular structure of the deletions was dramatically affected as determined by dimeric and monomeric profiles on size exclusion chromatography. It can be concluded that amino acids 428-444 (the C-terminal 17 amino acids) comprise an intersubunit binding domain that is required for tetramer formation of TPH, but that tetramer assembly is not essential for full enzymatic activity.",
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Carboxyl terminal deletion analysis of tryptophan hydroxylase. / Mockus, Susan M.; Kumer, Sean C.; Vrana, Kent.

In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, Vol. 1342, No. 2, 17.10.1997, p. 132-140.

Research output: Contribution to journalArticle

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