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 K d value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 lM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the K d value for isoform 4. Dopamine binds more much tightly, with K d values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in K d values of about 0.1 lM 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 K d values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH. Keywords: DOPA, dopomine, phosphorylation, regulation, tyrosine hydroxylase. The biosynthesis of the catecholamine neurotransmitters begins with the hydroxylation of tyrosine to form dihydroxyphenylalanine (DOPA). This step, catalyzed by the enzyme tyrosine hydroxylase (TyrH), is generally accepted to be the rate-limiting step in the pathway (Fitzpatrick 1999). The TyrH reaction is a monooxygenation, in that molecular oxygen is the source of the atom of oxygen incorporated into DOPA (Daly et al. 1968), whereas tetrahydrobiopterin serves as the source of the two electrons required for reduction of the other atom of oxygen to the level of water. Activation of oxygen for the reaction involves an active site non-heme iron atom; for catalysis, the iron atom must be in the ferrous form (Fitzpatrick 1989). In the presence of oxygen the iron is readily oxidized to the ferric form; tetrahydropterins can reduce the iron back to the active ferrous state (Ramsey et al. 1996). The active site containing this iron atom is a deep cleft in a catalytic domain of about 300 amino acids that is homologous to the catalytic domains of the two other pterindependent aromatic amino acid hydroxylases, phenylalanine hydroxylase and tryptophan hydroxylase (Grenett et al. 1987;Goodwill et al. 1997).All three enzymes also contain N-terminal regulatory domains. The sequences of thes...

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 three 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 substrates access; this conformation is favored by phosphorylation of Ser40.Tyrosine hydroxylase (TyrH) catalyzes the first and rate-limiting step of catecholamine biosynthesis, the conversion of tyrosine into dihydroxyphenylalanine, utilizing a tetrahydropterin as the source of electrons. The enzyme belongs to the small family of aromatic amino acid hydroxylases, which also includes phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) (1). All three enzymes play critical physiological roles; PheH is responsible for catabolism of excess phenylalanine in the diet, while TrpH is the first and rate-limiting enzyme in serotonin biosynthesis. The mammalian forms of these enzymes are homotetramers (2-4) in which each monomer contains a regulatory domain of 100-150 amino acids at the N-terminus and a larger catalytic domain of around 350 amino acids at the Cterminus (5-9). The homologous (5) catalytic domains contain all of the residues required for catalysis and for substrate specificity (10), while the regulatory domains exhibit low levels of sequence identity (5,11). Structures have been determined for the catalytic domains of all three enzymes (12)(13)(14), but the only regulatory domain with an available structure is that of PheH *Address correspondence to: Paul F. Fitzpatrick Department of Biochemistry, MC 7760 University of Texas Health Science Center at San Antonio San Antonio, TX 78229-3900 fitzpatrick@biochem.uthscsa.edu ph: 210-567-8264; fax: 210-567-8778. 1 Abbreviations used: TyrH, rat tyrosine hydroxylase; PheH, phenylalanine hydroxylase; TrpH, tryptophan hydroxylase; PKA, protein kinase A. Supporting Information Available:The effects of dopamine and phosphorylation on the kinetics of deuterium incorporation into all of the peptic peptides for TyrH. This material is available free of charge via the Internet at http://pubs.ac...

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.

There was an error in Table 4 of the above article which was published in J. Neurochem. 90, pp 970-978. The values for K d were given in lM, however, this should have been nM. Furthermore, incorrect reference to footnotes were cited. The publishers apologise for any inconvenience caused.