Identification by hydrogen/deuterium exchange of structural changes in tyrosine hydroxylase associated with regulation.

The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by 3 orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrate access; this conformation is favored by phosphorylation of Ser40.

Effects of ligands on the mobility of an active-site loop in tyrosine hydroxylase as monitored by fluorescence anisotropy.

Fluorescence anisotropy has been used to monitor the effect of ligands on a mobile loop over the active site of tyrosine hydroxylase. Phe184 in the center of the loop was mutated to tryptophan, and the three native tryptophan residues were mutated to phenylalanine to form an enzyme with a single tryptophan residue in the mobile loop. The addition of 6-methyl-5-deazatetrahydropterin to the enzyme resulted in a significant increase in the fluorescence anisotropy. The addition of phenylalanine did not result in a significant change in the anisotropy in the presence or absence of the deazapterin. The K(d) value for the deazapterin was unaffected by the presence of phenylalanine. Qualitatively similar results were obtained with apoenzyme, except that the addition of phenylalanine led to a slight decrease in anisotropy. Frequency-domain lifetime measurements showed that the distribution of lifetimes was unaffected by both the amino acid and deazapterin. Frequency-domain anisotropy analyses were consistent with a decrease in the motion of the sole tryptophan in the presence of the deazapterin. This could be modeled as a decrease in the cone angle for the indole ring of about 12 degrees . The data are consistent with a model in which binding of a tetrahydropterin results in a change in the conformation of the surface loop required for proper formation of the amino acid binding site.

Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms.

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.