EPR and 1H-NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors.
The paramagnetic iron at the active site of highly purified, catalytically active phenylalanine hydroxylase was studied by EPR at 3.6 K and one-dimensional 1H-NMR spectroscopy at 293 K. The EPR-detectable iron of the bovine enzyme was found to be present as a high-spin form (S = 5/2) in different ligand field symmetries depending on medium conditions (buffer ions) and the presence of ligands known to bind at the active site. At 3.6 K and in phosphate buffer, the paramagnetic iron is coordinated in an environment of rhombic symmetry (g = 4.3), whereas Tris buffer favours an environment of axial ligand field symmetry (g = 6.7, 5.3 and 2.0). The latter axial type of signals resembles those observed at g = 7.0, 5.2 and 1.9 for the enzyme in phosphate buffer when L-noradrenaline is added as an active-site ligand (inhibitor). The same proportion of iron that coordinates to L-noradrenaline seems to be reduced by the pterin cofactor and participate in catalysis. Experimental evidence is presented that Tris inhibits the enzyme by interacting with the enzyme-bound ferric iron and decreases its rate of reduction by the tetrahydropterin cofactor. Preincubation with dithiothreitol also inhibits the enzyme activity and prevents the reduction of its catalytically active ferric iron by pterin cofactors as well as binding of catecholamines to the enzyme. 1H-NMR spectroscopy revealed that the substrate (L-phenylalanine) and L-noradrenaline bind close to the paramagnetic iron, and that the catecholamine displaces the substrate from its binding at the active site. The results support our recently proposed model for the cooperative binding of inhibitor and substrate at the active site [Martínez, A. et al. (1990) Eur. J. Biochem. 193, 211-219].
Martínez
A
A
Department of Biochemistry, University of Bergen, Norway.
Andersson
K K
KK
Haavik
J
J
Flatmark
T
T
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006859:Hydrogen; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008670:Metals / Q000032:analysis; D009638:Norepinephrine / Q000494:pharmacology; D010651:Phenylalanine Hydroxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D014325:Tromethamine / Q000494:pharmacology
D008670:Metals; D014325:Tromethamine; D006859:Hydrogen; D010651:Phenylalanine Hydroxylase; D004229:Dithiothreitol; D009638:Norepinephrine