Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosyn thesis of serotonin. in the rabbit, TPH exists as a tetramer of four identi cal 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic do main. Previous studies demonstrated that within the carboxyl-terminus of TP H, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hy drophobic repeat identified within the regulatory domain of TPH (residues 2 1-41) may also be involved in macromolecular assembly. To test this hypothe sis, a series of amino-terminal deletions (N Delta 15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chr omatography. The amino-terminal deletion N Delta 15, upstream from the 4,3- hydrophobic repeat, was capable of forming tetramers. However, when a porti on of the 4,3-hydrophobic repeat was deleted (N Delta 30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete r emoval of the 4,3-hydrophobic repeat (N Delta 41) rendered the enzyme incap able of forming tetramers; a monomeric form predominated. In addition, a do uble-point mutation (V28R-L31R) was created in the hydrophobic region of th e enzyme. The introduction of two arginines (R) at positions 28 and 31 resp ectively, in the helix disrupted the native tetrameric state of TPH. Accord ing to size-exclusion chromatography analysis, the double-point mutant (V28 R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is infor mation within the amino-terminus that is necessary for tetramer formation o f TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.