Intersubunit binding domains within tyrosine hydroxylase and tryptophan hydroxylase

Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT) belongs to the aromatic amino acid hydroxylase superfamily, which includes phenylalanine hydroxylase (PAH) an d tyrosine hydroxylase (TH). The crystal structures for both PAH and TH hav e been reported, but a crystallographic model of TPH remains elusive. For t his reason, we have utilized the information presented in the TH crystal st ructure in combination with primary sequence alignments to design point mut ations in potential structural domains of the TPH protein. Mutation of a TH salt bridge (K170E) was sufficient to alter enzyme macromolecular assembly . We found that the disruption of the cognate intersubunit dimerization sal t bridge (K111-E223) in TPH, however, did not affect the macromolecular ass embly of TPH. Enzyme peaks representing only tetramers were observed with s ine exclusion chromatography. By contrast, a single-point mutation within t he tetramerization domain of TPH (L435A) was sufficient to disrupt the norm al homotetrameric assembly of TPH. These studies indicate that, although th e proposed salt bridge dimerization interface of TH is conserved in TPH, th is hypothetical TPH intersubunit binding domain, K111-E223, is not required for the proper macromolecular assembly of the protein. However, leucine 43 5 within the tetramerization domain is necessary for the proper macromolecu lar assembly of TPH. (C) 2000 Wiley-Liss, Inc.