Effects of phosphorylation on binding of catecholamines to tyrosine hydroxylase: Specificity and thermodynamics

As the catalyst for the rate-limiting step in the biosynthesis of the catec holamine neurotransmitters, the activity of tyrosine hydroxylase is tightly regulated. A principle means of posttranslational regulation is reversible phosphorylation of serine residues in an N-terminal regulatory domain. Pho sphorylation of serine 40 has been shown to have a large effect on the rate constant for dissociation of dopamine and a much smaller effect on that fo r DOPA [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980- 8986]. To determine the structural basis for the differences in affinity an d to further test the validity of the previously proposed model for regulat ion, the effects of phosphorylation of serine 40 on the affinities for a se ries of catechols have been determined. The affinities of the unphosphoryla ted enzyme vary by 3 orders of magnitude due to differences in the rates of dissociation. The highest affinities are found with catecholamines which l ack a carboxylate. The affinities of the phosphorylated enzyme show a much smaller range. In the case of binding of dihydroxyphenylalanine, the decrea se in affinity upon phosphorylation is due primarily to a decrease in the e nthalpy of the interaction. Based upon these results, a structural model fo r the effect of phosphorylation is proposed.