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Phenylalanine hydroxylase is an enzyme which catalyzes the conversion of L-phenylalanine to L-tyrosine (Tyr), the rate-limiting step in the oxidative degradation of phenylalanine, using the co-factor tetrahydrobiopterin (BH4) and molecular oxygen. PheOH is abundant in the liver that converts as much as 75% of ingested phenylalanine. Human phenylalanine hydroxylase (PAH) is the gene encoder for PheOH located on human chromosome 12. Mutation of PAH cause phenylketonuria (PKU), an autosomal recessive metabolic disorder, leading to severe forms of mental retardation. Unconverted phenylalanine gathered in the body and brain also caused mental retardation.
Amino acids are the building block of proteins. Twenty amino acids are commonly found in nature. Nine of these are essential to human that must be included in the diet because they cannot be synthesized from other precursors. These essential amino acids are phenylalanine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan and valine. Though these are essential to human, their amount in bloodstream should still be controlled. And so enzymes control the amount of amino acids into the bloodstream.
Phenylalanine (Phe), one of the essential amino acid into humans, when accumulated into the body can lead to neurological damage, while depletion of stores of this essential amino acid may lead to decrease in protein synthesis. Phenylalanine hydroxylase (PheOH) is a major enzyme responsible for Phe serum levels and disposal.
Phenylalanine Hydroxylase Structure
Phenylalanine hydroxylase is a tetrimetric enzyme. The four subunits of PheOH is comprised of three distinct domains: an N-terminal regulatory domain that contains an autoregulatory sequence (ARS), a central catalytic domain, and a short C-terminal tetramerisation domain(2). The N-terminal ARS function to control the activity of the enzyme. The central catalytic consists of 13 alpha helices and 8 beta strands located deep within the protein. The tetramerisation domain consists of 2 beta strands that form a long helix. 2-7 alpha helices wrap around one another forms coiled-coil motif which makes strong links to hold the enzyme together. Each subunits contain its own active site to catalyze the reaction of the enzyme.
Figure 1: Structure of Phenylalanine Hydroxylase
PheOH enzyme is related to tyrosine hydroxylase (TryOH) and tryptophan hydroxylase (TrpOH) according to FusettiÂ et al. (1998) because they found out many biochemical and physical similarities between the three enzymes. They share similarities in their domains but only differ in catalytic domain. "The absence of significant structural differences in the active site of PheOH and TyrOH is not surprising because a similar low substrate binding specificity is observed for the catalytic domains in steady-state kinetic analysis." (FusettiÂ et al. 1998)(1). The catalytic domain of PheOH is contains a hydrophobic cage location for hydroxylation.
Mechanism of catalysis and Kinetics of Reaction
Phenylalanine hydroxylase (PheOH) is a tetrametric and iron-dependent enzyme which catalyzes the conversion of L-phenylalanine (Phe) to L-tyrosine (Tyr) using tetrahydrobiopterin (BH4) as a reducing agent and molecular oxygen as an oxidizing agent.
The reaction of the enzyme mechanism proceeds a steps for its conversion to tyrosine: formation of a Fe(II)-O-O-BH4Â bridge prior to heterolytic cleavage, and the hydroxylation of phenylalanine by ferryl oxo intermediate. Formation of a Fe(II)-O-O-BH4Â bridge prior to heterolytic cleavage remain controversial, two models were proposed based on the number of water molecules and proximity of the iron to the pterin cofactor during the catalysis.
Figure 2: Formation Fe(IV)=O
According to one model, as a resonance hybrid of Fe2+O2Â and Fe3+O2- an iron dioxygen is initially formed and so the activated O2 attacks BH4 for a transition state by charge separation for the formation of Fe(II)-O-O-BH4 bridge. Although molecular oxygen is the source of both oxygen atoms used for the hydroxylation of pterin ring and phenylalanine, this model still predicts a different mechanism because once Fe(II)-O-O-BH4 bridge is finally formed, the O-O bond will be broken to Fe(IV)=O and 4a-hydroxytetrahydrobiopterin throughÂ heterolytic cleavage (Fig.2)
Figure 3: Hydroxylation of Phenylalanine to Tyrosine
Unproductive consumption of BH4 and formation of H2O2 is now the result because of the oxidation of BH4Â cofactor and hydroxylation of phenylalanine was decoupled. As initially proposed in radical intermediate in the analysis of tyrosine and tryptophan hydroxylases, it was suggested that the reaction will proceed to a cationic intermediate that requires Fe(IV)=O be coordinated to a water ligand rather than a hydroxo group(4). Cationic intermediate undergoes NIH shift that then tautomerizes to form tyrosine product (Fig 3)(3). The pterin cofactor undergoes hydration to form BH2 which will be reduced to BH4.
Mode of Regulation
Activation by the substrate phenylalanine is a reversible process that involves all subunits of the tetramer. The enzyme substrate can bind at two sites in its mode of regulation. It can bind in the active site where it undergoes hydroxylation, and in the allosteric site where it regulates PheH activation(2). Both local and global conformational changes in the enzyme were induced by the binding of the substrate. BH4 cofactor displaced at the active site which also releases its interaction with autoregulatory sequence (ARS) exposing it to phosphorylation. Phosphorylation acts as a conformational switch that facilitates Phe-driven PheH activation. Increase in the rate of PheH phosphorylation makes the activation of the enzyme. Cofactor BH4 binding also inhibits phosphorylation by contrast.
Associated Diseases and Applications
Defects in the DNA of the individual are usually refered to as inborn errors of matabolism. Mutations leading to deficiencies in enzymes that catalyze reaction of amino acids may lead to severe forms of mental retardation. Phenylalanine, phenylpyruvate, phenyllactate, and phenylacetate are all stored in the blood and urine. As an evidence, phenylpyruvate causes mental retardation because ot interferes the conversion of pyruvate to acetyl-CoA in the brain. Acetyl-CoA is an important intermediate in many biochemical reactions. Accumulation of these products in the brain results in the osmotic imbalance. Osmotic balance is when water flows into the brain cells and they expand in size until they crush in the developing brain which results to inability to develop normally.
One common example of inborn errors of metabolism is phenylketonuria. Phenylketonuria (PKU) is a genetic disorder that is characterized by the inability of the body to break down protein. Protein cannot properly convert phenylalanine so levels of the amino acid increase throughout the body. A normal blood phenylalanine level is about 1 mg/dl and high level of Phe in the blood ranges 6 to 80mg/dl. High levels of Phe in the blood cause significant brain problems when accumulate in the brain.
Another disease that is related to deficiency of phenylalanine breakdown enzyme is hyperphenylalaninemia. Hyperphenylalaninemia means elevated blood phenylalanine. It happens due to the lack of another enzyme important to the process of the enzyme(5). It is usually described as group of disorders.
Causes of hyperphenylalaninemia and PKU are inherited. This means that the 'carrier', a person with one trait for the disorder, inherited it one from each parent. Infants with PKU have early symptoms such irritability, an eczema-like rash, vomitting and a mousy odor to the urine. Later symptoms will be microcephaly (small head), severe brain problems and seizures.
For early identification and implementation of treatment, new born babies undergo screening test wherein a few drops of blood are obtained by a small pick on the heel placed on a card, and then sent for measurement(5).
This disease requires diet. Phenylalanine must be limited to the amount needed for protein synthesis. High protein food such as milk, fish, cheese, meat, and poultry should be avoided. Fruits and vegetables are recommended. Aspartame, a sweetener in drinks, is also implemented because it has a component phenylalanine. A substitute for this is Alatame, contains alanine instead of phenylalanine. It retain the benefits of aspartame without the dangers associated with phenylalanine.