Tyrosine hydroxylase activity is regulated by two distinct dopamine-binding sites

Tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline, is regulated acutely by feedback inhibition by the catecholamines and relief of this inhibition by phosphorylation of serine 40. Phosphorylation of serine 40 abolishes the binding of dopamine to a high affinity (K(D) <4nM) site on tyrosine hydroxylase, thereby increasing the activity of the enzyme. We have found that tyrosine hydroxylase also contains a second low affinity (K(D) = 90nM) dopamine binding site, which is present in both the non-phosphorylated and the serine 40-phosphorylated forms of the enzyme. Binding of dopamine to the high affinity site decreases Vmax and increases the Km for the cofactor BH(4), while binding of dopamine to the low affinity site regulates tyrosine hydroxylase activity by increasing the Km for BH(4). Kinetic analysis indicates that both sites are present in each of the four human tyrosine hydroxylase isoforms. Dissociation of dopamine from the low affinity site increases tyrosine hydroxylase activity 12-fold for the non-phosphorylated enzyme and 9-fold for the serine 40-phosphorylated enzyme. The low affinity dopamine binding site has the potential to be the primary mechanism responsible for the regulation of catecholamine synthesis under most conditions.