Phenylalanine hydroxylase is an enzyme, which is necessary for the catalysis of phenylalanine to tyrosine. This catalysis is realized by hydroxylation of the aromatic side chain of phenylalanine. The enzyme needs tetrahydrobiopterin, BH4 a pteridine cofactor, and a non-heme iron for catalysis. In the reaction, molecular oxygen is cleaved. One oxygen atom is incorporated in BH4. The other oxygen atom is incorporated in phenylalanine. The reaction is shown in the following schema.
This reaction is the bottleneck in the metabolic pathway, which degrades phenylalanine. Mutations in the gene of PAH can cause the metabolomic disorder phenylketonuria. The structure of phenylalanine hydroxylase is very well reviewed. In this article we want to summarize the results in order to acquired more insight into the effect of mutations of the gene of phenylalanine hydroxylase.
In this illustration the binding pockets are very obvious. The red Fe atoms in the binding pockets mark their positions.
It is assumed, that the reaction takes place in three steps:
- formation of a Fe(II)-O-O-BH4 bridge
- heterolytic cleavage of the O-O bond to yield the ferryl oxo hydroxylating intermediate Fe(IV)=O
- attack on Fe(IV)=O to hydroxylate phenylalanine substrate to tyrosine.
Especially the formation of the Fe(II)-O-O-BH4 bridge is strongly discussed. Therefore most crystallographic experiments focus on the catalytic domain, which contains the BH4 and the Fe-atom.
Phenylalanine hydroxylase consists of three domains:
- a regulatory N-terminal domain (residues 1-117)
- the catalytic domain (residues 118-427)
- a C-terminal domain (residues 428-453) responsible for oligomerization of identical monomers
Regulatory N-terminal domain
Phenylalanine hydroxylase is regulated by a kind of allosteric activation. Phenylalanine needs to be at a certain concentration and after a short lag time the enzyme starts to catalyze the phenylalanine. This behaviour can be explained by the N-terminal domain, which consists of the residues 1-117. The regulatory nature of this domain is caused by its structural flexibility.
Buried active site
Hydrogen/deuterium exchange analysis shows, that the interface between the regulatory and the catalytic domain is increasingly exposed to the solvent by the allosteric binding of phenylalanine. This can be explained by a global altering of the conformation of the enzyme. This change contains an exposing of the active site to the solvent.
This argumentation is supported by kinetic studies. These studies show an initially low rate of tyrosine formation for full-length PheOH. This lag time is not observed, for a truncated PheOH lacking the N-terminal domain or if the full-length enzyme is pre-incubated with Phe. Deletion of the N-terminal domain also eliminates the lag time while increasing the affinity for Phe by nearly two-fold.
An additional source of regulation is the Ser16. The phosphorylation of Ser16 does not alter enzyme conformation but does reduce the concentration of Phe required for allosteric activation.
It is supposed that the active site consists of an open and spacious pocket lined primarily by hydrophobic residues. This pocket contains also three glutamic acid residues, two histidines, and a tyrosine, which are critical for pterin- and iron-binding. Contradictory evidence exists about the coordination state of the ferrous atom and its proximity to BH4 within the active site. Fe(II) is coordinated by water, His285, His290 and Glu330. Inclusion of a Phe analogue in the crystal structure both changes 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 towards 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 analysis suggests that all coordinated water molecules are forced out of the active site during the catalytic cycle while BH4 becomes directly coordinated to iron. This discrepancy will be important for determining the exact mechanism of PAH catalysis.
C-terminal oligomerization domain
The C-terminal oligomerization domain contains the residues 428-453. The procaryotic PAH is monomeric. The eukaryotic PAH exists in an equilibrium between homotetrameric and homodimeric forms. The dimerization interface is composed of symmetry-related loops that link identical monomers. 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. A domain swapping mechanism has been proposed to mediate formation of the tetramer from dimers shifting equilibrium towards the tetrameric form. In this mechanismin C-terminal alpha-helices mutually alter their conformation around a flexible C-terminal five-residue hinge region to form a coiled-coil structure.
Both the homodimeric and homotetrameric forms of PheOH are catalytically active. But the two exhibit differential kinetics and regulation. In addition to reduced catalytic efficiency, the dimer does not display allosteric activation by phenylalanine. This suggests that phenylalanine allosterically regulates PAH by influencing the dimer-dimer interaction.
Phenylalanine hydroxylase has been the protein of the month (January 2005).