Identification of substrate orienting and phosphorylation sites within tryptophan hydroxylase using homology-based molecular modeling

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. The inherent instability of TPH has prevented a crystallographic structure from being resolved. For this reason, multiple sequence alignment-based molecular modeling was utilized to generate a full -length model of human TPH. Previously determined crystal coordinates of tw o highly homologous proteins, phenylalanine hydroxylase and tyrosine hydrox ylase, were used as templates. Analysis of the model aided rational mutagen esis studies to further dissect the regulation and catalysis of TPH. Using rational site-directed mutagenesis, it was determined that Tyr235 (Y235), w ithin the active site of TPH, appears to be involved as a tryptophan substr ate orienting residue. The mutants Y235A and Y235L displayed reduced specif ic activity compared to wild-type TPH (approximate to 5 % residual activity ). The K-m of tryptophan for the Y235A (564 mu M) and Y235L (96 mu M) mutan t was significantly increased compared to wild-type TPH (42 mu M). In addit ion, kinetic analyses were performed on wild-type TPH and a deletion constr uct that lacks the amino terminal autoregulatory sequence (TPH N Delta 15). This sequence in phenylalanine hydroxylase (residues 19 to 33) has previou sly been proposed to act as a steric regulator of substrate accessibility t o the active site. Changes in the steady-state kinetics for tetrahydrobiopt erin (BH,) and tryptophan for TPH N Delta 15 were not observed. Finally, it was demonstrated that both Ser58 and Ser260 are substrates for Ca2+/calmod ulin-dependent protein kinase II. Additional analysis of this model will ai d in deciphering the regulation and substrate specificity of TPH, as well a s providing a basis to understand as yet to be identified polymorphisms. (C ) 2000 Academic Press.