Characterization of the human tryptophan hydroxylase isoforms

Tryptophan hydroxylase (TPH) catalyzes the hydroxylation of tryptophan to L-5-hydroxytryptophan, which is the first and rate-limiting step in the biosynthesis of 5-hydroxytryptamine (serotonin). Serotonin acts as a hormone and neurotransmitter in a variety of tissues and is involved in a wide range of physiological functions. Dysregulation of the level of serotonin is associated with a variety of physiological and psychiatric disorders. TPH exists in two homologous isoforms; TPH1 and TPH2. Isoform 1 is primarily expressed in the peripheral tissues, while isoform 2 is mainly found in the central nervous system. As the rate-limiting enzymes in the synthesis of serotonin, TPH1 and TPH2 play vital roles in the serotonergic systems of the peripheral tissues and central nervous system, respectively.

The active form of TPH contains iron(II) and catalyzes tryptophan hydroxylation utilizing 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and molecular oxygen. The TPH isoforms are members of an enzyme subfamily of iron(II)-containing mono-oxygenases, referred to as the aromatic amino acid hydroxylases. The family also includes phenylalanine hydroxylase and tyrosine hydroxylase. These homologous enzymes all form homotetramers, in which each subunit comprises an N-terminal regulatory domain, a highly conserved catalytic domain, and a C-terminal tetramerization domain.

In this dissertation, both human TPH isoforms were under investigation. Of the isoforms, TPH2 is less characterized in literature due to low purification quantities caused by its inherent instability and tendency to aggregate. To overcome this challenge, three variants of human TPH2 with deletion mutations of entire or parts of domains were expressed, purified, and examined. Removal of the C-terminal tetramerization domain resulted in TPH variants which could be purified in quantities sufficient for enzymatic characterization. However, this variant suffered from a high inactivation rate. Upon further removal of the N-terminal regulatory domain, a significant decrease in rate of inactivation was observed. This observation renders the regulatory domain the main source of instability. To overcome the inherent instability of the regulatory domain, differential scanning fluorimetry was used to identify stabilizing ligands. In absence of ligands the unfolding was continuous and polyphasic. However, in presence of tryptophan or phenylalanine the unfolding shifted to apparent two-state, indicative of increased thermostability and monodispersity. The shift in unfolding behavior was accompanied by a ligand concentration-dependant increase in transition temperature. This thermostabilizing effect was confirmed by a significant decrease in inactivation rate. Analytical gel filtration revealed that in the presence of the regulatory domain, the TPH2 variant resides in a monomer-dimer equilibrium. With the addition of phenylalanine a significantly shift towards dimer was observed explaining the ligand-induced increase in thermostability. These results led to the addition of phenyalanine in the purification buffer solutions which significantly increased the purification yields.

Very little is known about the catalytic mechanism of TPH and most of the knowledge in literature stem from extrapolations of results obtained for the other members of the enzyme subfamily. In this dissertation, results are presented that demonstrate that the steady-state kinetic mechanism of the catalytic domain of human TPH1 follows a hybrid Ping Pong-ordered mechanism. In this mechanism, the reaction can either occur through a Ping Pong or a sequential mechanism depending on the concentration of tryptophan, and substrate inhibition occurs via competitive inhibition of BH4 binding. The kinetic study also revealed that the isoforms display very different kinetic properties despite their high sequence identity. One of the major differences is that TPH1 is substrate inhibited, while TPH2 is not. By scrutinizing the crystal structures of the isoforms, it was found that differences reside in the orientation of a loop lining the active site. Point mutations were conducted within this loop, and significant changes in the kinetic parameters of the mutant TPH1 variants were observed. Molecular dynamics simulations revealed that the substrate inhibition mechanism occurs through a closure of the BH4 binding pocket upon tryptophan binding, and that the active site loop is involved in this mechanism by propagating structural changes from the tryptophan binding site to the BH4 binding pocket.