Stabilization of tryptophan hydroxylase 2 by l-phenylalanine-induced dimerization.

Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full-length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rch TPH2; regulatory and catalytic domain, NΔ47-rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14- and 11-fold higher half-lives compared to rch TPH2 and NΔ47-rch TPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rch TPH2 and NΔ47-rch TPH2 are found to shift from polyphasic to apparent two-state by the addition of l-Trp or l-Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47-rch TPH2 reside in a monomer-dimer equilibrium which is significantly shifted toward dimer in the presence of l-Phe. The dimerizing effect induced by l-Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half-lives of rch TPH2 and NΔ47-rch TPH2. Addition of l-Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2.

Experimentally calibrated computational chemistry of tryptophan hydroxylase: trans influence, hydrogen-bonding, and 18-electron rule govern O2-activation.

Insight into the nature of oxygen activation in tryptophan hydroxylase has been obtained from density functional computations. Conformations of O(2)-bound intermediates have been studied with oxygen trans to glutamate and histidine, respectively. An O(2)-adduct with O(2)trans to histidine (O(his)) and a peroxo intermediate with peroxide trans to glutamate (P(glu)) were found to be consistent (0.57-0.59mm/s) with experimental Mössbauer isomer shifts (0.55mm/s) and had low computed free energies. The weaker trans influence of histidine is shown to give rise to a bent O(2) coordination mode with O(2) pointing towards the cofactor and a more activated O-O bond (1.33A) than in O(glu) (1.30A). It is shown that the cofactor can hydrogen bond to O(2) and activate the O-O bond further (from 1.33 to 1.38A). The O(his) intermediate leads to a ferryl intermediate (F(his)) with an isomer shift of 0.34mm/s, also consistent with the experimental value (0.25mm/s) which we propose as the structure of the hydroxylating intermediate, with the tryptophan substrate well located for further reaction 3.5A from the ferryl group. Based on the optimized transition states, the activation barriers for the two paths (glu and his) are similar, so a two-state scenario involving O(his) and P(glu) is possible. A structure of the activated deoxy state which is high-spin implies that the valence electron count has been lowered from 18 to 16 (glutamate becomes bidentate), giving a "green light" that invites O(2)-binding. Our mechanism of oxygen activation in tryptophan hydroxylase does not require inversion of spin, which may be an important observation.

Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms.

Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed, purified and the kinetic properties have been studied and are compared. Substrate inhibition by tryptophan is observed for isoform 1 but not for isoform 2. Large differences are observed in the K (m,tetrahydrobiopterin) values for the two isoforms, being >10 times larger for isoform 1 compared to isoform 2.

A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization.

Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.