Carboxyl terminal deletion analysis of tryptophan hydroxylase.
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 51007 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 delta8, C delta12 and C delta17) 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.
Mockus
S M
SM
Program in Neuroscience, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1083, USA.
Kumer
S C
SC
Vrana
K E
KE
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D004252:DNA Mutational Analysis; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014365:Tryptophan Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014365:Tryptophan Hydroxylase