CRYSTALLOGRAPHIC ANALYSIS OF THE HUMAN PHENYLALANINE-HYDROXYLASE CATALYTIC DOMAIN WITH BOUND CATECHOL INHIBITORS AT 2.0 ANGSTROM RESOLUTION

The aromatic amino acid hydroxylases represent a superfamily of struct urally and functionally closely related enzymes, one of those function s being reversible inhibition by catechol derivatives. Here we present the crystal structure of the dimeric catalytic domain (residues 117-4 24) of human phenylalanine hydroxylase (hPheOH), cocrystallized with v arious potent and well-known catechol inhibitors and refined at a reso lution of 2.0 Angstrom. The catechols bind by bidentate coordination t o each iron in both subunits of the dimer through the catechol hydroxy l groups, forming a blue-green colored ligand-to-metal charge-transfer complex. Ic. addition, Glu330 and Tyr325 are identified as determinan t residues in the recognition of the inhibitors. In particular, the in teraction with Glu330 conforms to the structural explanation for the p H dependence of catecholamine binding to PheOH, with a pK(a) value of 5.1 (20 degrees C). The overall structure of the catechol-bound enzyme is very similar to that of the uncomplexed enzyme (rms difference of 0.2 Angstrom for the C alpha atoms). Most striking is the replacement of two iron-bound water molecules with catechol hydroxyl groups. This change is consistent with a change in the ligand field symmetry of the high-spin (S = 5/2) Fe(III) from a rhombic to a nearly axial ligand f ield symmetry as seen upon noradrenaline binding using EPR spectroscop y [Martinez, A., Andersson, K. K., Haavik, J., and Flatmark, T. (1991) fur. J. Biochem. 198, 675-682]. Crystallographic comparison with the structurally related rat tyrosine hydroxylase binary complex with the oxidized cofactor 7,8-dihydrobiopterin revealed overlapping binding si tes for the catechols and the cofactor, compatible with a competitive type of inhibition of the catechols versus BH4. The comparison demonst rates some structural differences at the active site as the potential basis for the different substrate specificity of the two enzymes.