Tryptophan hydroxylase 2 (TPH2) hydroxylates L-tryptophan to L-5-hydroxytryptophan (5-HTP) the first and rate-limiting step of serotonin (5-HT) synthesis in the mammalian brain. Some mutations in the Tph2 gene reducing TPH2 activity are associated with hereditary depressive disorders. The P447R substitution in the mouse TPH2 molecule reduces its thermal stability in vitro and its activity in the brain. The effects of iron ions on thermal stability in vitro and the activity in the brain of the mutant TPH2 were investigated. In the in vitro experiment effects of 0.01, 0.05, and 0.2 mM of FeSO₄ and FeCl₃ on the enthalpy (ΔH) and Gibbs free energy (ΔG) of thermal denaturation of the mutant TPH2 extracted from the midbrain of Balb/c mice were assayed. All FeSO₄ concentrations and 0.05 and 0.2 mM concentrations of FeCl₃ increased these thermodynamic characteristics of the mutant TPH2. Repeated (for 7 days) intramuscular administration of Fe(III) hydroxide dextran complex (15 and 30 mg/kg/day) increased TPH2 activity in the hippocampus, but not in the midbrain in Balb/c mice. Repeated (for 7 days) intramuscular administration of Fe(III) hydroxide dextran complex (15 and 30 mg/kg/day) together with thiamine (8 mg/kg/day) and cyanocobalamin (0.8 mg/kg/day) increased TPH2 activity in the hippocampus, while 30 mg/kg of Fe(III) hydroxide dextran also increased the enzyme activity in the midbrain in Balb/c mice. These results are the first evidence for chaperone-like effects of iron ions on thermal stability in vitro and activity in the brain of the mutant TPH2.

Tyrosine hydroxylase (TH) catalyzes the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is the rate-limiting step in dopamine (DA) synthesis in the brain. Certain mutations in the human TH gene are associated with severe neurological and psychiatric disorders. The c.886C>T mutation in the mouse Th gene, which results in the R278H substitution, decreases TH activity in the mouse midbrain. Here, we compared the molecular characteristics of the wild-type (TH-278R) and mutant (TH-278H) recombinant forms of TH. Both recombinant proteins were expressed in Escherichia coli BL21 cells and purified by immobilized metal affinity chromatography using Ni-NTA agarose resin. TH activity was assayed by HPLC, and the thermal stability of the recombinant proteins was assessed by differential scanning fluorimetry. The R278H substitution markedly decreased V_max and increased the K_M for the cofactor 6,7-dimethyltetrahydropterin (DMPH₄), but had no effect on the K_M for the substrate L-tyrosine. The substitution did not affect the T₅₀ of thermal inactivation but markedly increased the T_m of thermal unfolding of the TH-278H protein. The substitution decreased the IC₅₀ values for dopamine and 1,2-dihydroxybenzene several-fold, both of which contain a catechol moiety that is also present in L-DOPA. These results indicate that the R278H substitution alters the sensitivity of TH to catechol-containing compounds. Although direct evidence is lacking, endogenous L-DOPA produced during the reaction may interact differently with the catalytic center of the TH-278H mutant than with that of the wild-type TH-278R.

Pharmacological chaperones (PCs)—small molecules that normalize the 3D structure of mutant protein molecules—are promising substances for pharmacological treatment of grave hereditary pathologies. In this study, possible chaperone-like effects of the Fe(II) with tris(1-pyrasolyl)methane complex, [Fe(TPM)₂]Cl₂, on the mutant 447R form of mouse tryptophan hydroxylase 2 (TPH2) in vitro and in vivo were investigated. The experiments were carried out on Balb/c mice homozygous for the mutant TPH2. This complex in concentrations of 0.01, 0.05 and 0.1 mM markedly increased the temperature (T₅₀) and free energy (ΔG) of the mutant TPH2 thermal denaturation in vitro. Seven intramuscular administrations of 30 and 60 mg/kg of [Fe(TPM)₂]Cl₂ markedly increased the TPH2 activity in the midbrain of Balb/c mice. This increase in the TPH2 activity was not accompanied with an increase in the Tph2 gene mRNA and TPH2 protein levels. It is the first demonstration of chaperone-like activity of [Fe(TPM)₂]Cl₂. This complex is a promising chemical for correction of genetic alterations in TPH2 and the associated hereditary psychic disorders.