CARBOXYL-TERMINAL DELETION ANALYSIS OF TRYPTOPHAN-HYDROXYLASE

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step ill the synthesis of serotonin and participates (in a non-rate-limiting fashio n) in melatonin biosynthesis. In rabbit, TPH exists as a tetramer of f our identical 51007 dalton (444 amino acids) protein subunits. An inte rsubunit 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 f or the assembly of tyrosine hydroxylase tetramers. Polymerase chain re action was utilized to create three TPH carboxyl terminal deletions (C Delta 8, C Delta 12 and C Delta 17) that sequentially remove members of the leucine heptad and 4-3 hydrophobic repeat. Each deletion and fu ll-length recombinant TPH was expressed in bacteria to obtain soluble enzyme extracts for subsequent activity and structural analysis. It wa s found that removal of 8, 12 or 17 amino acids from the carboxyl term inus of TPH did not significantly alter enzymatic activity when compar ed to full-length recombinant TPH. However, the macromolecular structu re of the deletions was dramatically affected as determined by dimeric and monomeric profiles on size exclusion chromatography. It can be co ncluded that amino acids 428-444(the C-terminal 17 amino acids) compri se an intersubunit binding domain that is required for tetramer format ion of TPH, but that tetramer assembly is not essential for full enzym atic activity. (C) 1997 Elsevier Science B.V.