Phenylketonuria is due to enzymatic dysfunction of phenylalanine hydroxylase. In the PAH Mutation Analysis Consortium Database (Nowacki 1997), there have been identified over 230 mutations of which many of them have been associated with varying degrees of severity in regards to phenotypes of PKU. Most diseases that involve enzyme dysfunction are usually as a result of a mutations which affect a direct location of catalysis. However, PKU is defined by its inborn error in metabolism as a protein folding disease. So, PKU is defined by its loss of function due to its destabilization (Gersting 2008). A majority of the mutations of PAH that result in the phenotype of PKU patients is based on mutations of the regulatory domain and very few mutations are located at the catalytic active site. The discussion of allostery has been observed as the mechanism by which these mutations affect PAH enzymatic activity.
A study by Gersting et al (2008) looked at the effect of 10 PAH mutations that have been correlated with BH4 responsiveness. The authors identified that although enzymatic activity was possible, unstable conformation due to the disruption of allostery could lead to the enzyme’s degradation. The authors looked at mutations in different locations on the regulatory domain and evaluated their effects. Two mutations, S310Y and R408W, which were located at the hydrophobic center of the protein, resulted in significant disruption of its structural activity ultimately leading to aggregation. Other mutations in the flexible regions of the enzyme such as, H170Q, P275L, P314S, and Y417H resulted in significant loss of enzymatic function as well. Although these mutations did not cause significant conformational changes in the enzyme, the mutations are involved in a network of amino acids that are involved in conformational stability and function of the enzyme. These mutations had direct affects on the efficiency of the allosteric interactions and cooperativity, but little effect on enzyme catalysis, thus suggesting their potential success with BH4 supplementation to encourage proper folding and enzyme stability. Other mutations at the regulatory domain gave more information to why such mutations resulted in PAH dysfunction. Carluccio et al. (2013) demonstrated a mechanism by which mutations in the regulatory domain could affect hydrophobic interactions inside the regulatory domain. The conformational motions in the wildtype demonstrate a dense network of connections suggesting that allosteric movement happens concertedly, while multiple mutations demonstrated less connections suggesting less effective allosteric movement of the enzyme, as demonstrated below (Carluccio 2013). These regulatory domain mutations can result in incorrect binding of the phenylalanine activator, resulting in poor phenylalanine activation, decreased allostery, and thus decrease in enzyme activity (Carluccio 2013).
Another hypothesis in regards to why these mutations result in PKU symptoms was outlined by Jaffe et al. (2013). They identified that phenylalanine activation at the ACT portion of the regulatory domain results in a shift of equilibrium between non-active dimers and tetramers to the active tetramer of PAH. The argument for the link of many mutations in PKU to the dysfunction of PAH is not associated with monomeric folding failures, but actually associated with the inability for the phenylalanine activated quaternary active tetramer to be formed due to instability of the binding or the affected amino acid networks that have been associated with the conformational change and the active oligomerization. This will thus shift the equilibrium back toward the inactive dimers and tetramers which have been demonstrated to form larger oligomers that form aggregates (Jaffe et al. 2013). When PAH aggregates and forms a pile of misfolded proteins or and aggresome, the aggresome is ubiquitinated which in the end results PAH degradation by the autophagolysosomes (Leandro 2011).
With over 500 plus mutations, pin-pointing one specific mechanism by which PAH mutations yield protein dysfunction is impossible. Protein dysfunction of PAH occurs by many different mechanisms. The main mechanism for enzyme dysfunction revolves around regulatory domain mutations that result in problems with allostery, phenylalanine activation, and cooperativity. These mutations that cause regulatory domain misfolding have led to inactive conformations of PAH.
Since the mechanism by which the mutation results in less effective PAH has been theorized, the consequences of PAH dysfunction need to be defined. The resultant biochemical problem is due to the accumulation of phenylalanine that results from the PAH dysfunction (Vockley 2014). This is due to the fact that phenylalanine is constitutively digested under most normal diets and since it cannot be converted into tyrosine it begins to build up in the blood and in the brain. Since there is a build-up of phenylalanine, phenylalanine undergoes decarboxylation and transamination chemistry to make phenylethylamine and phenylpyruvate respectively, resulting in the musty odor demonstrated by the Egeland children (Centerwall 2000). The compound pheylpyruvate in the patient’s urine, which is made via the transamination of phenylalanine, when reacted with ferric chloride resulted in the green color and thus the discovery of the disease.
The neuropathogenesis of PKU has not been explained by a direct mechanism, but a correlation between high phenylalanine concentrations in the blood and neurological phenotypes has been established. There are many potential theories for the multitude of neurological symptoms that are caused by this enzyme dysfunction. The first and most probable mechanism is believed that somehow, indirectly high concentrations of phenylalanine in the blood results in hypomyelination. PKU patients when untreated experience high levels of phenylalanine, hypomyelination in the forebrain, and decreased dopamine levels (Joseph 2003). Myelin is an active membrane that is essential for conduction of action potentials across axons. Hypomyelination as a result of this disease PKU is most likely caused by a downstream effect of high phenylalanine concentrations. One theory is that phenylalanine, which is a moderate inhibitor of HMG-CoA reductase, could prevent the synthesis of cholesterol which a major lipid utilized in the biosynthesis of the myelin sheath (Joseph 2003). Another theory for how hypomyelination might occur in the brain is that elevated phenylalanine concentrations in the blood will out-compete other large neutral amino acids across the blood brain barrier amino acid transporter (Pietz 1999). This would result in an accumulation of phenylalanine in the brain and a decrease in the amount of other large neutral amino acids in the brain resulting in overall decreased protein production in the brain, which could cause deleterious effects and the neurodegenerative pathology (Pietz 1999). Decreased levels of protein synthesis in the brain could also lead to decrease levels of enzymes that synthesize essential neurotransmitters in the brain (Joseph 2003). Loss of myelin could also lead to down-regulation of this neurotransmitter production (Joseph 2003). White matter lesions have also been associated with PKU (cannot get access to any of these articles).
Another interesting observation about downstream effects of phenylalanine accumulation in the blood includes its regulatory role in enzymes that are involved in oxidative stress. A potential theory for neurological pathology could be associated with phenylalanine-mediated oxidative stress. Moraes et al. (2013) determined that phenylalanine inhibited catalase activity while some of the phenylalanine derivatives produced in PKU, such as phenyllactic and phenylacetic acids stimulated superoxide mutase activity. This results in the formation of hydrogen peroxide and hydroxide radicals that can induce oxidative stress on the brain which could be associated with these neurological pathologies. Also, discovered by Moraes et al. (2014), phenylalanine accumulation has been associated with decreased glutathione levels in PKU patients. The authors analyzed the expression of enzymes involved in the metabolism and function of glutathione in order to discover the effects of elevated phenylalanine concentrations in PKU patients. Glutathione peroxidase, glucose-6-phosphate dehydrogenase, and glutathione reductase all demonstrated a decrease activity due to high phenylalanine concentrations in the blood. The antioxidant effect of glutathione is thus decreased in high phenylalanine blood concentrations suggesting oxidative stress causes for the neurological pathologies as well.