R278H Substitution Reduces Mouse Tyrosine Hydroxylase Activity and Increases Susceptibility to Catechol Inhibition
- 1Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, Russia
Abstract
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 Vmax and increased the KM for the cofactor 6,7-dimethyltetrahydropterin (DMPH4), but had no effect on the KM for the substrate L-tyrosine. The substitution did not affect the T50 of thermal inactivation but markedly increased the Tm 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.
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BibTeX
@article{alsalloum2026r278h,
title = {{R278H Substitution Reduces Mouse Tyrosine Hydroxylase Activity and Increases Susceptibility to Catechol Inhibition}},
author = {Ismail Alsalloum and Ekatherine I. Terentieva and Vitalii S. Moskaliuk and Sophia M. Antonova and Tatiana V. Ilchibaeva and Alexander V. Kulikov},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27209370},
url = {https://www.mdpi.com/1422-0067/27/20/9370},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - R278H Substitution Reduces Mouse Tyrosine Hydroxylase Activity and Increases Susceptibility to Catechol Inhibition
AU - Ismail Alsalloum
AU - Ekatherine I. Terentieva
AU - Vitalii S. Moskaliuk
AU - Sophia M. Antonova
AU - Tatiana V. Ilchibaeva
AU - Alexander V. Kulikov
PY - 2026
DA - 2026/10/21
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27209370
UR - https://www.mdpi.com/1422-0067/27/20/9370
AB - 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 Vmax and increased the KM for the cofactor 6,7-dimethyltetrahydropterin (DMPH4), but had no effect on the KM for the substrate L-tyrosine. The substitution did not affect the T50 of thermal inactivation but markedly increased the Tm 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.
SN - 1422-0067
ER -