History and Metabolic Context of Phenylketonuria

Phenylketonuria (PKU) is an autosomal recessive genetic disorder that is inherited due to a contribution of the genetic disorder gene from each parent. Phenylketonuria is due to a variety of mutations to the gene of the liver enzyme phenylalanine hydroxylase (PAH) (Vockley 2014). Patients that have phenylketonuria demonstrate symptoms that have been associated with mental retardation (Centerwell 2000). Phenotypes differ depending on the severity of the PKU. Different PAH mutations result in different levels of PAH dysfunction. PKU severity is usually correlated with the concentration of phenylalanine in the blood caused by the mutation; however, genotypes do not always result in a distinct PKU phenotype (Pey 2003). Severe PKU symptoms, when not treated, consist of seizures, serious behavioral and social problems, stunted growth, decreased IQ levels, increased occurrences of eczema, mental retardation, fair skin, and an overall musty odor as well as in the urine (Vockley 2014). Most of these phenotypes are associated with the more severe forms of PKU with the most severe phenotypes of mental retardation. The normal phenylalanine levels in the blood of non-PKU patients is below 120 umol/L, but the more severe causes, known as “classic PKU” are defined by a phenylalanine blood concentration above 1,200 umol/L (Vockley 2014). Elevated phenylalanine concentrations have been associated with many other biochemical changes in patients with PKU, however the mechanism by which elevated phenylalanine concentrations yields mental retardation is unclear.  Hypotheses revolving around the indirect linkage between elevated phenylalanine levels and hypomyleination could potentially elucidate the missing link from genotypic mutation and PAH dysfunction to PKU phenotypes. Although severe phenotypes have been noticed prior to discovery, new born screening methods such as Guthrie’s test have led to early newborn detection of PKU. This early detection coupled with immediate implementation of a low phenylalanine diet has significantly lowered the occurrence of severe PKU phenotypes. Keeping a low phenylalanine concentration has resulted in decreased physical neurological deficits and prevents the neurological symptoms that arise due to elevated levels of phenylalanine in the blood and in the brain. Other treatments are in the works and will be discussed later.

Dr. Folling who discovered Phenylketonuria, the first inborn error in metabolism.
Dr. Folling who discovered Phenylketonuria, the first inborn error in metabolism.

As demonstrated by Siegried and Willard Centerwall (2000), the discovery of PKU and treatment of PKU started with a professor, Dr. Asbjorn Folling, and young couple that had two kids with symptoms of mental retardation. The couple Borgny and Harry Egeland was in search of help for discovering the cause of their kid’s mental disability. The couple noticed an unusual smell in their son’s and daughter’s urine and were wondering if the odor could be connected to their children’s phenotypes. Dr. Asbjorn Folling conducted the usual testing on urine for albumin, blood, and pus, but they all came back normal. However, a urine test looking for the presence of ketones in the blood resulted in positive results. The test conducted involved using 10% aqueous ferric chloride solution on the urine sample changing the color of the solution to an unusual dark green color. Dr. Asbjorn Folling was able to crystallize the ketone-containing compound in the urine and determined its melting point to be 155oC. He was able to determine that this ketone in fact contained a benzene ring, in which he hypothesized to be the presence of phenylpyruvic acid. Dr. Folling then proceeded to conduct the ferric chloride screening on other patients experiencing the same phenotypes as the Egeland children. 8 others were identified to have the presence of phenylpyruvic acid in the urine. He was able to determine from pedigree analysis that this disease was based on autosomal recessive inheritance. This discovery of PKU was the first ever inborn error in metabolism that was connected to mental retardation phenotypes (Centerwell 2000) .

 

 

The result of the ferric chloride test for ketones that demonstrated the presence of phenylpyruvic acid in PKU patient's urine.
The result of the ferric chloride test for ketones that demonstrated the presence of phenylpyruvic acid in PKU patient’s urine.

Shortly following the discovery of PKU, a simple phenylalanine diet was demonstrated to have significant advantageous effects on the neurological phenotype of many PKU patients (Blainey 56). Their results suggested that implementation of a low phenylalanine diet soon after prognosis resulted in a decrease in phenylalanine concentrations in the blood, improved neurological function (as a result of IQ testing), and overall prevention of the neurological damage associated with PKU (Blainey 56). Around the same time period, Jervis et al. (Centerwell 2000) was able to identify that PKU was a result of enzymatic deficiency, which was identified to be the enzymatic dysfunction of phenylalanine hydroxylase, which was later determined to be caused by a variety of mutations.  In the 1960’s, Dr. Robert Guthrie was able to identify a new method of infant screening for PKU that identifies the concentration of phenylalanine in the blood. This test was also used to monitor the effectiveness of the low phenylalanine diet utilized by patients with PKU (Guthrie 1963). The test radically changed the effectiveness of PKU diagnosis and was useful at monitoring the effectiveness of PKU diet.  Over the 60 or so years since PKU’s discovery, over 500 mutations of phenylalanine hydroxylase have been associated with the emergence of symptoms of PKU. An effort to identify and consolidate the mutations into a database was conducted in 1997 under the PAH Mutation Analysis Consortium Database (Nowacki 1997). Nowacki et al. were able to identify 238 mutations that were associated with PKU, with the mutations varying in severity, and hyperphenylalanemia. This database identified all of the mutations to date and their exact location on the 12q24.1 locus, also known as the locus that is transcribed to make phenylalanine hydroxylase.

 

The locus for PAH is at 12q24.1 locus. This demonstrates all of the possible mutations that were known as of 1997 when PAH Mutation Analysis Consortium Database was consolidated.
The locus for PAH is at 12q24.1 locus. This demonstrates all of the possible mutations that were known as of 1997 when PAH Mutation Analysis Consortium Database was consolidated.

 

Both children have PKU but the child on the right has been implementing a phenylalanine restricted diet while the boy on the left has not.
Both children have PKU but the child on the right has been implementing a phenylalanine restricted diet while the boy on the left has not.

 

 

 

 

 

As mentioned prior, the PKU phenotypic pathology is due to mutations in the enzyme PAH. Although, understanding this enzyme’s dysfunction is essential for understanding the biochemical causes of PKU and potential treatments, it is essential that we understand the biochemistry of a successfully functioning wild type PAH to elucidate the biochemical deviations that cause the pathology of PKU. Phenylalanine hydroxylase is a homotetrameric enzyme that consists of three main domains. These three domains include the N-termini regulatory domain, the catalytic domain and the oligomerization domain at the C-termini (Gertsing 2008). Below, Fusetti et al. (1998) determined the three dimensional protein structure of the active homotetramer of PAH.

 

This is a 3D crystal structure of the wildtype human PAH, which was crystallized by Fusetti et al. (1998).
This is a 3D crystal structure of the wildtype human PAH, which was crystallized by Fusetti et al. (1998).

In catalysis, the enzyme utilizes a non-heme bound iron, a tetrahydrobiopterin (BH4) cofactor, molecular oxygen, and its natural substrate phenylalanine (Roberts 2012). The enzyme binds non-heme iron at the active site using a facial 2-His-1-Glu arrangement (Roberts 2012). This iron has been hypothesized to be the essential mediator in catalysis of molecular oxygen addition to BH4 and to the aromatic amino (Fitzpatrick 2003). In order to have a better understanding on catalysis, Roberts et al. looked at the kinetics of the catalytic domain separate from its regulatory domain to formulate a possible enzymatic model for how the enzyme functions (Roberts 2012). Roberts et al. determined the following kinetic mechanism for PAH by conducting a unique set of single turnover reactions which were globally simulated by the use of KinTek Explorer to extract microscopic rates of the enzymatic reaction. The following kinetic mechanism below was proposed for the enzymatic reaction of PAH below.

 

The catalytic kinetic mechanism determined by Roberts et al. in 2012.
The catalytic kinetic mechanism determined by Roberts et al. in 2012.

The kinetic mechanism that was elucidated from their findings demonstrated that the PAH first binds to its cofactor BH4. There exist two BH4 specific binding events that can occur that can led to either a productive or a dead end BH4-PAH complex. Roberts et al. (2012) theorized that the binding of BH4 in its active or open conformation results, in an accessible active site for its phenylalanine substrate. This “productive” binding event also suggests potential ternary complexation that is observed in active PAH. However, a dead-end BH4 binding has been recognized in which the binding of BH4 inhibits the catalysis of the enzyme by this closed formation binding, resultant from the associated Tyr138 loop closure that makes phenylalanine binding at the active site impossible. This inhibitory BH4-E complex must dissociate in order for PAH to become active again. Such inhibitory effects caused by BH4 have been suggested structurally with BH4’s association with Ser23 of the regulatory domain stabilizing this inactive form of PAH. Also, the inhibiting Phe-E complex was demonstrated in this paper as the first of its kind. The authors suggest that phenylalanine in high enough concentrations can completively bind to the BH4 binding site, resulting in loss of catalysis. Next, once the productive PAH complex is made it is able to incorporate phenylalanine into the active site forming the complex with the PAH, BH4, and its substrate phenylalanine. The next step following was defined by the binding of the molecular oxygen at the active site which is bound to the iron (II) atom. The actual enzymatic catalytic reaction was too fast to be observed by limitations of the stopped-flow apparatus, but potential theories have been formulated for how the reaction might carry out (Roberts 2012). The enzymatic reaction has been defined to be split into two main components: first the formation of the Fe(IV)O intermediate which conducts the hydroxylation (which has been observed) and the oxygen transfer to the para position of the aromatic phenylalanine substrate. Previous research explored in close homologs of PAH, like tryptophan hydroxylase and tyrosine hydroxylase, have suggested the presence of the Fe(II)-peroxypterin intermediate and the Fe(IV)O intermediate.  The theorized kinetic reaction demonstrated below outlines the theory by which each partial enzymatic reaction occurs. In the creation of the Fe(IV)O intermediate,  molecular oxygen is bound to Iron (II) donates using two electron chemistry to react with BH4 in a flavin-like manner in order to generate the hydroxylated BH4, which subsequently is eliminated releasing water resulting in its quinoid tautomer, and the Fe(IV)O reactive intermediate.  The quinoid tautomer then gets reduced by NADH at another enzyme’s active site to regenerate BH4. The Fe(IV)O species in then utilized to hydroxylate the phenylalanine at the para position. The Fe(IV)O is then attacked by the aromatic ring of the phenylalanine in order to conduct an electrophilic aromatic substitution which results in the hydroxylated phenylalanine, also known as the enzyme’s product tyrosine. An interesting characteristic that has been observed for this reaction is the presence of a hydride shift from the C4 position (para position) to the adjacent carbon that is electrophilic. This hydride shift or transfer is known as a NIH shift, in the second image below.

 

Formation of Iron (IV) oxo species.
Formation of Iron (IV) oxo species.

 

Hydroxylation of phenylalanine to form tyrosine and a NIH shift is observed during this reaction.
Hydroxylation of phenylalanine to form tyrosine and a NIH shift is observed during this reaction.

After the enzymatic reaction has been completed, the tyrosine product then was released, which was determined by the authors to be the rate limiting step, which defined the kcat of PAH. In conclusion, most of the kinetic mechanism has been determined for PAH, and a slightly understanding of how BH4 inhibits PAH has been elucidated through this mechanism and our understanding of structure.

 

Understanding catalysis is essential for describing PAH functionality, however PAH dysfunction which results in PKU has been associated with allosteric effects of PAH. PAH works based on many different forms of regulation. For instance, phenylalanine acts as an allosteric activator to PAH, while as demonstrated above BH4 can act as an inhibitor. This enzyme can also be regulated by phosphorylation events, as demonstrated by H/D exchange and mass spectrometry. The activation by phosphorylation of Ser16 in PAH by cAMP-dependent protein kinase A, has been demonstrated to cause a similar conformational change to that of phenylalanine, but the change is less significant (Li 2013).  Although this phosphorylation event demonstrated less of an impact on activation, it was demonstrated that less concentration of phenylalanine is required upon phosphorylation in order to activate PAH and shift the enzymatic conformation of PAH into its active homotetramer (Li 2013). Also, as demonstrated prior BH4 binding can inhibit the enzymatic activity of PAH by forcing a closed loop conformation that blocks the active site preventing the ability for phenylalanine to bind at its active site. The most well studied regulatory mechanism of PAH is its activation by phenylalanine.  The N-terminal regulatory domain of PAH contains as ACT domain which acts as an allosteric regulator for the entire quaternary strucutre of PAH by its activation via phenylalanine activator which induces a large conformational change upon phenylalanine binding (Carluccio 2013). Thus, phenylalanine when bound at the interface of the ACT portion of the regulator domain and another ACT portion of a regulatory domain is associated with conformational change of the regulatory domain that promotes the active conformation of the PAH to carry out its enzymatic reaction. This activation by phenylalanine binding to the regulatory domain leads to allostery which results in a shift of oligomerization of PAH into its active homotetramer form, instigating catalysis (Carluccio 2013). Another study supported these claims about phenylalanine activation, but also demonstrated that this phenylalanine-activated PAH demonstrates cooperativity and that phenylalanine activitation leads to positive cooperativity in BH4 binding (Gersting 2010). Another important recent study on allostery demonstrated the how phenylalanine activation drives catalysis. Jaffe et al. demonstrated the mechanism of conformational change caused phenylalanine activation results in a draw of the equilibrium of the inactive tetramer and dimer to its active tetramer, which results in exposure of catalytic active site and thus enzymatic activity. This allosteric regulation if dysfunctional is essential for understanding why PKU occurs (Jaffe 2013).

 

Allosteric model of homotetramer with the 4mer on the right being the active conformation of the enzyme.
Allosteric model of homotetramer with the 4mer on the right being the active conformation of the enzyme.