Carboxyl terminal deletion analysis of tryptophan hydroxylase

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

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

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

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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