1. A Molecular View of Phenylketonuria By: Sindhu Kilakkathi

2. What is Phenylketonuria (PKU)? Autosomal recessive genetic disorder (Incidence: 1/10,000 newborns) Mutation of Phenylalanine hydroxylase (PAH) gene on chromosome 12 1 –Lack of PAH, defective PAH, insufficient BH 4 Normal PAH BH 4

3. What is Phenylketonuria (PKU)? Autosomal recessive genetic disorder (Incidence: 1/10,000 newborns) Mutation of Phenylalanine hydroxylase (PAH) gene on chromosome 12 1 –Lack of PAH, defective PAH, insufficient BH 4 Mutated PAH BH 4

4. Effects of PAH Enzymopathy Accumulation of Phe  elevated blood and urine Phe levels Decreased levels of Tyr  lack of catecholamines (necessary for development) and melanin BH 4

5. Symptoms of PKU Severe mental retardation, brain damage, seizures, mousy odor in skin and hair, lack of pigmentation, eczema, IQ of <50, aggressive behavior, negative mood –Reduced dopamine levels –hypomyelination 5 Variability in expressivity More prevalent in certain ethnic populations (e.g. Turkish- 1/2600) 1 http://www.dshs.state.tx.us/newborn/over_pku.shtm

6. Diagnosis PKU first detected with FeCl 3 test (phenylpyruvate + FeCl 3  green urine) 2 Now Guthrie test is used (4 mg/dL) Prenatal testing (DNA probes) 1 http://www.flickr.com/photos/ozewiezewozewiezewallakristallix/2632833781/

7. Implications for Carriers of PKU 1.5% population are heterozygotes Appear phenotypically normal, but have lower levels of PAH and higher blood levels of Phe Definitive test: measure kinetics of disappearance of injected Phe 2 ?

8. Treatment Low Phe diet started soon after birth (restricted protein and dairy consumption) 3 Supplements- BH 4 (Kuvan and Phenoptin) Phenylalanine ammonia lyase (enzyme substitution) Gene therapy (viral vectors to introduce functional PAH gene into liver cells) 1

9. Understanding the Biochemistry of PKU Root of the Problem: –Mutations in PAH [PDB ID: 2PAH] Cause of the Symptoms: –Interaction of Phe with Large Neutral Amino Acids (LNAAs) at the Blood Brain Barrier (BBB) [PDB ID: 2DH2] Treatment: –How PAL can treat PKU [PDB ID: 1Y2M]

10. A Closer Look at PAH Tetramer Monomer 2 identical alpha subunits – cyan 2 identical beta subunits – red RegulatoryDomain

11. Mutations of PAH 400+ mutations of PAH gene that cause PKU Most frequent mutation- R408W Tryptophan substituted for arginine

12. An Examination of LNAAs LNAA carriers transport LNAAs across BBB LAT1 transporter encoded for by SLC3A2 and SLC3A5 genes in 4F2hc 4F2hc (CD98hc)- multifunction type II membrane glycoprotein Phe blocks transport of Tyr and Trp Acetone

13. Treatment with PAL Another enzyme that can catabolize Phe

14. PAL Structure Homotetrameric 4 identical monomers Overlap of the 4 subunits

15. Issues with PAL in Treatment Accessible PAL trypin and chymotrypsin cleavage sites- Arg123 and Tyr 110 Conjugate with Polyethylene gycol (PEG) to increase efficacy So far there have not been major side effects

16. References 1. Williams RA, Mamotte CD, Burnett JR. Phenylketonuria: an inborn error of phenylalanine metabolism. Clin Biochem Rev. 2008; 29(1):31-41. 2. Berg, Jeremy Mark., John L. Tymoczko, and Lubert Stryer. Biochemistry. 6th ed. New York: W.H. Freeman, 2007. 673-74. Print. 3. Michals-Matalon K. Developments in phenylketonuria. Topics Clin Nutr. 2001;16:41–50. 4. Dyer CA. Pathophysiology of phenylketonuria. Ment Retard Dev Disab Res Rev. 1999;5:104–12. 5. Erlandsen H, Stevens RC. 1999. The structural basis of phenylketonuria. Mol Genet Metab 68: 103-125.