The<unk>  monomer (51.9 kDa) consists of three distinct domains: a regulatory N-terminal domain (residues 1–117) that contains a<unk> e-binding ACT subdomain, the catalytic domain (residues 118–427), and a C-terminal domain (residues 428–453) responsible for oligomerization of identical monomers. Extensive crystallographic analysis has been performed, especially on the pterin- and iron-coordinated catalytic domain to examine the active site. The structure of the N-terminal regulatory domain has also been determined, and together with the solved structure of the homologous tyrosine hydroxylase C-terminal tetramerization domain, a structural model of tetrameric<unk>  was proposed. Using X-ray crystallography, the structure of full-length rat<unk>  was determined experimentally and showed the auto-inhibited, or resting-state form of the enzyme. The resting-state form (RS-PAH) is architecturally distinct from the activated form (A-PAH). A full-length structure of A-PAH is currently lacking, but the<unk> e stabilized ACT-ACT interface that is characteristic of A-PAH has been determined and a structural model of A-PAH based on SAXS analysis has been proposed. Model of the active site of<unk>  bound to BH4, ferrous, and a<unk> enylalanine analogue. (from PDB 1KW0)<unk> enylalanine analogue, BH4, iron, Fe(II)-coordinated His and Glu residues Catalytic domain Solved crystal structures of the catalytic domain indicate that the active site consists of an open and spacious pocket lined primarily by hydrophobic residues, though three glutamic acid residues, two histidines, and a tyrosine are also present and iron-binding. Contradictory evidence exists about the coordination state of the ferrous atom and its proximity to BH4 within the active site. According to crystallographic analysis, Fe(II) is coordinated by water, His285, His290, and Glu330 (a 2-his-1-carboxylate facial triad arrangement) with octahedral geometry. Inclusion of a<unk> e analogue in the crystal structure changes both iron from a six- to a five-coordinated state involving a single water molecule and bidentate coordination to Glu330 and opening a site for oxygen to bind. BH4 is concomitantly shifted toward the iron atom, although the pterin cofactor remains in the second coordination sphere. On the other hand, a competing model based on NMR and molecular modeling analyses suggests that all coordinated water molecules are forced out of the active site during the catalytic cycle while BH4 becomes directly coordinated to iron. As discussed above, resolving this discrepancy will be important for determining the exact mechanism of<unk>  catalysis. N-terminal regulatory domain The regulatory nature of the N-terminal domain (residues 1–117) is conferred by its structural flexibility. Hydrogen/deuterium exchanges analysis indicates that allosteric binding of<unk> e globally alters the conformation of<unk>  such that the active site is less occluded as the interface between the regulatory and catalytic domains is increasingly exposed to solvent. This observation is consistent with kinetic studies, which show an initially low rate of tyrosine formation for full-length<unk> . This lag time is not observed, however, for a truncated<unk>  lacking the N-terminal domain or if the full-length enzyme is pre-incubated with<unk> e. Deletion of the N-terminal domain also eliminates the lag time while increasing the affinity for<unk> e by nearly two-fold; no difference is observed in the Vmax or Km for the tetrahydrobiopterin cofactor. Additional regulation is provided by Ser16;<unk> osphorylation of this residue does not alter enzyme conformation but does reduce the concentration of<unk> e required for allosteric activation. This N-terminal regulatory domain is not observed in bacterial<unk> s but shows considerable structural homology to the regulatory domain of<unk> osphogylcerate dehydrogenase, an enzyme in the serine biosynthetic pathway. Tetramerization domain Prokaryotic<unk>  is monomeric, whereas eukaryotic<unk>  exists in an equilibrium between homotetrameric and homodimeric forms. The dimerization interface is composed of symmetry-related loops that link identical monomers, while the overlapping C-terminal tetramerization domain mediates the association of conformationally distinct dimers that are characterized by a different relative orientation of the catalytic and tetramerization domains (Flatmark, Erlandsen). The resulting distortion of the tetramer symmetry is evident in the differential surface area of the dimerization interfaces and distinguishes<unk>  from the tetramerically symmetrical tyrosine hydroxylase. A domain-swapping mechanism has been proposed to mediate formation of the tetramer from dimers, in which C-terminal alpha-helixes mutually alter their conformation around a flexible C-terminal five-residue hinge region to form a coiled-coil structure, shifting equilibrium toward the tetrameric form. Although both the homodimeric and homotetrameric forms of<unk>  are catalytically active, the two exhibit differential kinetics and regulation. In addition to reduced catalytic efficiency, the dimer does not display positive cooperativity toward L-Phe (which at high concentrations activates the enzyme), suggesting that L-Phe allosterically regulates<unk>  by influencing dimer-dimer interaction.
Phenylalanine hydroxylase
Aromatic L-amino acid decarboxylase
Catechol-O-methyltransferase
Tyrosine hydroxylase
Dopamine beta-hydroxylase
Glutamate dehydrogenase
Monoamine oxidase B
Monoamine oxidase