Conformational Properties and Stability of Tyrosine Hydroxylase Studied by Infrared Spectroscopy

Aurora Martínez, Jan Haavik, Torgeir Flatmark, José Luis R. Arrondo, Arturo Muga
1996 Journal of Biological Chemistry  
The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% ␣-helix, 35% ␤-extended structures (including ␤-sheet), 14% ␤-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance
more » ... ity and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in ␣-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure. Tyrosine hydroxylase (TH, 1 EC 1.14.16.2) is a non-heme iron and tetrahydrobiopterin-dependent enzyme that catalyzes the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DO-PA), the first and rate-limiting step in the biosynthesis of catecholamines (1, 2). The catalytic activity of TH seems to be short-term regulated by feedback inhibition by catecholamines and by reversal of this inhibition by phosphorylation (1, 3). Until the recent cloning and overexpression of TH from different species (4 -6), the source of the enzyme has mostly been the bovine adrenal medulla or pheochromocytoma (PC12) cells. However, these enzymes are isolated partially inhibited by catecholamines bound at the active site and are activated severalfold by phosphorylation of Ser-40 by the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (7-9). This activation has been related to the increased dissociation rate of catecholamines from the active site on phosphorylation (3) . Human TH exists as several different isoforms generated by alternative splicing of pre-mRNA (10, 11); isoform 1 (hTH1) is the most abundant species in the adrenal medulla and in the substantia nigra of the brain (10). When expressed in Escherichia coli, human TH isoforms are isolated as homogeneous tetrameric apoenzymes composed of identical 56-59-kDa subunits (12) that are rapidly activated (up to 40-fold) by binding of 1 atom of Fe(II) per subunit (4). The recombinant human enzymes are inhibited by catecholamines that chelate iron at the active site, and this inhibition is partially reversed by phosphorylation of Ser-40 by cAPK (3, 12, 13) . The three-dimensional structure of TH remains to be determined, and the potential conformational changes associated with the activation by phosphorylation and inhibition by catecholamines are not characterized. It has been postulated that phosphorylation induces a conformational change that decreases the thermostability of the enzyme (14, 15). However, the influence of the enzyme-bound catecholamines on these effects was not considered. In the present work, we have studied the secondary structure and stability of recombinant hTH1, with special reference to the effects of its activation by Fe(II) and phosphorylation by cAPK and its inhibition by dopamine. As experimental techniques, we have used infrared (IR) spectroscopy and limited tryptic proteolysis. MATERIALS AND METHODS Dopamine and deuterium oxide were from Sigma. Human TH isoform 1 (hTH1) was expressed in E. coli and purified to homogeneity as described (4, 12). Buffers were passed through a Chelex-100 ion-exchange resin to avoid iron contamination. The enzyme preparations used in this study contained 0.02 Ϯ 0.01 atoms of iron/subunit, as determined by atomic absorption spectrometry, and was considered to represent the apoenzyme (apo-hTH1). The holoenzyme, Fe(II)-hTH1, was prepared by incubation of apo-hTH1 with equimolar concentrations of ferrous ammonium sulfate for 5 min at 20°C (4, 16), and the dopamine-Fe(III)-hTH1 complex was obtained by the incubation of Fe(II)-hTH1 with equimolar concentrations of dopamine (16). The concentration of purified recombinant enzyme was determined by the absorbance at 280 nm (⑀ 1% ϭ 10.4 cm Ϫ1 ) at neutral pH (7) . The catalytic subunit of cAPK was purified from bovine heart as described (17) . Phosphorylated Fe(II)-hTH1 at Ser-40 was prepared from the phosphorylated apo-hTH1 as described for the nonphosphorylated form of the enzyme (see above). Phosphorylation of hTH1-apo-hTH1 (25 mM) was incubated with 0.
doi:10.1074/jbc.271.33.19737 pmid:8702679 fatcat:o3ocwvymd5fulhf6hn2pij6giy