Amino acids are the building blocks of proteins, and their sequence in a protein determines the 3-dimensional shape and ultimately the function of that protein.
Of the twenty amino acids commonly found in nature, nine are considered to be “essential” in humans, because they cannot be synthesised from other precursors, and therefore must be included in the diet.
These essential amino acids are phenylalanine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan and valine. Even though these amino acids are deemed essential, their relative amounts in the bloodstream must be tightly controlled.
For example, with phenylalanine (Phe), the accumulation of Phe can lead to neurological damage, while too rapid a degradation of Phe will lead to the depletion of stores of this essential amino acid and consequently a decrease in protein synthesis.
Enzymes control the levels of amino acids in the bloodstream, which can fluctuate widely due to dietary intake. In the case of Phe, the degradative enzyme phenylalanine hydroxylase is the major enzyme for Phe disposal, and is responsible for serum Phe levels.
The Protein Alphabet
The proteins that make up the skin, muscle, hair, bones and other organs in your body are primarily composed of a set of 20 building blocks, called amino acids. Amino acids are the alphabet in the protein language: when combined in a specific order, they make up meaningful structures (proteins) with varied and specific functions. Amino acids have distinct shapes, sizes, charges and other characteristics. Many amino acids are synthesized in your body from breakdown products of sugars and fats, or are converted from other amino acids by the action of specific enzymes. However, a few of them, called essential amino acids, cannot be synthesized or converted in your body and have to be obtained from the food you eat. Phenylalanine is one such essential amino acid. It is closely related to another amino acid, tyrosine, which just has an additional hydroxyl (OH) group. Liver cells contain an enzyme called phenylalanine hydroxylase, which can add this group and convert phenylalanine to tyrosine. Thus as long as this enzyme is functional and there is a reasonable supply of phenylalanine, tyrosine can be synthesized in your body and does not have to be included in the food that you eat.
Four molecules of phenylalanine hydroxylase interact to form a tetramer, which is the functional unit for this enzyme. Each molecule in the tetramer is organized into three domains: a regulatory domain, a catalytic domain where the enzyme activity resides and a tetramerization domain that assembles four chains into the tetramer.
At the heart of each catalytic domain is an iron ion that plays an important role in the enzyme action. A model structure of the complete enzyme tetramer is shown here.
This is composed of two PDB files: PDB entry 2pah, which includes the structure of the catalytic and tetramerization domains of the enzyme, and PDB entry 1phz, which includes the regulatory domain flexibly attached to the catalytic domain.
Surprisingly, we usually get too much phenylalanine in our diet, and this can cause problems. Normally, phenylalanine hydroxylase regulates the clearance of about 75% of the excess phenylalanine from our body by converting it to tyrosine. But in 1934, a Norwegian doctor, Asbjorn Folling, showed that the urine of two of his young mentally handicapped patients had a high level of phenylalanine.
These were the first diagnosed cases of phenylketonuria. Within the next few years it was shown that the absence or malfunction of phenylalanine hydroxylase leads to this serious genetic disorder. However, it was not until the early 1950s that this information was put to use.
A child suffering from phenylketonuria was treated with a diet low in the amino acid phenylalanine, showing that many of the symptoms of this disease could be reversed if treated early. Presently, in many places, newborn children are routinely screened for phenylketonuria and placed on a low phenylalanine diet if diagnosed.
As these children grow up, they are also recommended to avoid the sweetener Aspartame, since it contains phenylalanine. In a few cases, lack of the tetrahydrobiopterin, an essential cofactor for the enzyme, or an inability to regenerate it can also lead to phenylketonuria.
These cases may be treated with tetrahydrobiopterin supplements.
An Aromatic Alliance
The tyrosine synthesized by the action of phenylalanine hydroxylase is required for the synthesis of various neurotransmitters that act on the nervous system and also control key functions like respiration and heart rate.
During the synthesis of these neurotransmitters, tyrosine is further hydroxylated by the enzyme tyrosine hydroxylase. Interestingly, phenylalanine hydroxylase (PDB entry 1pah, shown on the left) and tyrosine hydroxylase (PDB entry 2toh, shown on the right) are structurally and functionally very similar to each other and also to tryptophan hydroxylase (PDB entry 1mlw).
The last enzyme acts on the related amino acid tryptophan. All three enzymes use an iron ion, function as tetramers and have similar domain architecture. Since phenylalanine, tyrosine and tryptophan all have aromatic ring structures, these hydroxylases are together called aromatic amino acid hydroxylases.
Exploring the Structure
The hydroxylation of phenylalanine requires free oxygen and a helper molecule (cofactor), tetrahydrobiopterin. Although the exact mechanism of the enzyme action is not known, it is clear that the cofactor interacts with a few conserved residues in the enzyme and the primary function of the iron ion is to stabilize this cofactor.
The backbone structure of the catalytic domain of phenylalanine hydroxylase is shown here (PDB entry 1j8u) with the tetrahydrobiopterin cofactor (colored green) and the iron ion (yellow sphere).
During the course of the reaction the cofactor loses two of its hydrogen atoms to form dihydrobiopterin (as can be seen in PDB entry 1dmw). Yet another enzyme acts on dihydrobiopterin to restore the original form of the cofactor, which is then used in another cycle of hydroxylation.
Mutations in phenylalanine hydroxylase can block the conversion of phenylalanine to tyrosine. Several hundred mutations have been documented in this enzyme. Many of these mutations destroy the enzyme activity and lead to phenylketonuria.
few examples are shown here in red. These mutations disrupt the interaction of the enzyme with either the cofactor or the iron ion, reducing or stopping the enzyme activity. Other serious mutations (not shown here) are found in amino acids that stabilize the structure of the enzyme, on the face that forms the enzyme tetramer or in regions that interact with the regulatory domain.
PheH controls the rate of Phe catabolism, and indirectly the rate of protein and neurotransmitter biosynthesis. In addition, the metabolites of Phe degradation are toxic to the developing brain; therefore, PheH must be under strict regulatory control in eukaryotes, despite fluctuations in dietary intake.
PheH is regulated in three main ways: BH4 cofactor inhibition, substrate activation, and phosphorylation.
In addition to being an essential co-substrate for catalysis, the BH4 cofactor helps regulate the enzyme. The binding of the BH4 cofactor at the active site results in a PheH‑BH4 complex that inhibits both substrate activation by Phe and phosphorylation of the enzyme, possibly by pulling the ARS region further into the active site to block it. Furthermore, BH4 may be important for enzyme stability, shielding the active site against damage by reactive oxygen species.
Activation by its substrate, Phe, is a reversible process that involves all subunits of the tetramer. Phe can bind at two sites, the active site where it undergoes hydroxylation, and an allosteric site where it regulates PheH activation.
The binding of Phe induces both local and global conformational changes in the enzyme, which displace the BH4 cofactor bound at the active site. The displacement of the BH4 cofactor releases its interaction with the ARS, which displaces the ARS from the active site as well as exposing it to phosphorylation.
Phosphorylation of PheH by cAMP-dependent protein kinase (PKA) involves a single serine residue, Ser16, in the N-terminal ARS region. Phosphorylation of Ser16 induces a conformational change in the ARS region and in the Phe-binding region of the active site.
These conformational changes permit greater accessibility of Phe to the active site by displacing the ARS, as well as increasing the binding affinity of the active site for Phe. In this way, phosphorylation acts as a conformational switch that facilitates Phe-driven PheH activation.
The activation of PheH is a cooperative process, where the subsequent binding of Phe serves to increase the rate of PheH phosphorylation, driving the activation of the enzyme. By contrast, the binding of the BH4 cofactor inhibits phosphorylation.