R278H Substitution Reduces Mouse Tyrosine Hydroxylase Activity and Increases Susceptibility to Catechol Inhibition

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.