Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization

Abstract

Tryptophan hydroxylase 2 ( TPH 2) 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 TPH 2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH 2 (rc h TPH 2; regulatory and catalytic domain, NΔ47‐rc h TPH 2; truncation of 47 residues in the N terminus of rc h TPH 2, and c h TPH 2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. c h TPH 2 displayed 14‐ and 11‐fold higher half‐lives compared to rc h TPH 2 and NΔ47‐rc h TPH 2, 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 rc h TPH 2 and NΔ47‐rc h TPH 2 are found to shift from polyphasic to apparent two‐state by the addition of l ‐Trp or l ‐Phe. Analytical gel filtration revealed that rc h TPH 2 and NΔ47‐rc h TPH 2 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 rc h TPH 2 and NΔ47‐rc h TPH 2. Addition of l ‐Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of h TPH 2.