CONFORMATIONAL PROPERTIES AND STABILITY OF TYROSINE-HYDROXYLASE STUDIED BY INFRARED-SPECTROSCOPY - EFFECT OF IRON CATECHOLAMINE BINDING ANDPHOSPHORYLATION/

The conformation and stability of recombinant tetrameric human tyrosin e hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis, Its secondary structure was estima ted to be 42% alpha-helix, 35% beta-extended structures (including bet a-sheet), 14% beta-turns, and 10% nonstructured conformations. Additio n of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significa ntly modify its secondary structure. However, an increased thermal sta bility and resistance to proteolysis, as well as a decreased cooperati vity in the thermal denaturation transition, was observed for the liga nd-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 it s overall conformation but allowed on iron binding a conformational ch ange characterized by an increase (about 10%) in alpha-helix and prote in stability. Our results suggest that the conformational events invol ved in TH inhibition by catecholamines are mainly related to modificat ions of tertiary and quaternary structural features. However, the comb ined effect of iron binding and phosphorylation. which activates the e nzyme, also involves modifications of the protein secondary structure.