H. Erlandsen et al., CRYSTALLOGRAPHIC ANALYSIS OF THE HUMAN PHENYLALANINE-HYDROXYLASE CATALYTIC DOMAIN WITH BOUND CATECHOL INHIBITORS AT 2.0 ANGSTROM RESOLUTION, Biochemistry (Easton), 37(45), 1998, pp. 15638-15646
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.