Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability.
A significant share of patients with phenylalanine hydroxylase (PAH) deficiency benefits from pharmacological doses of tetrahydrobiopterin (BH(4)), the natural PAH cofactor. Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH(4) treatment might be transmitted by correction of protein misfolding. To elucidate the molecular basis of functional impairment in PAH deficiency, we investigated the impact of ten PAH gene mutations identified in patients with BH(4)-responsiveness on enzyme kinetics, stability, and conformation of the protein (F55L, I65S, H170Q, P275L, A300S, S310Y, P314S, R408W, Y414C, Y417H). Residual enzyme activity was generally high, but allostery was disturbed in almost all cases and pointed to altered protein conformation. This was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding--with particular impact on the regulatory domain--observed in most variants. Three-dimensional modeling revealed the involvement of functionally relevant amino acid networks that may communicate misfolding throughout the protein. Our results substantiate the view that PAH deficiency is a protein-misfolding disease in which global conformational changes hinder molecular motions essential for physiological enzyme function. Thus, PKU has evolved from a model of a genetic disease that leads to severe neurological impairment to a model of a treatable protein-folding disease with loss of function.
Department of Molecular Pediatrics, Children's Research Center, Dr. von Hauner Children's Hospital, Ludwig Maximilians University, 80337 Munich, Germany.
Søren W
SW
Gersting
Kristina F
KF
Kemter
Michael
M
Staudigl
Dunja D
DD
Messing
Marta K
MK
Danecka
Florian B
FB
Lagler
Christian P
CP
Sommerhoff
Adelbert A
AA
Roscher
Ania C
AC
Muntau
D000284:Administration, Oral; D000494:Allosteric Regulation; D000592:Amino Acid Metabolism, Inborn Errors; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D001708:Biopterins / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000627:therapeutic use; D020134:Catalytic Domain; D003198:Computer Simulation; D019281:Dimerization; D019286:Endopeptidase K / Q000494:pharmacology; D004795:Enzyme Stability; D005260:Female; D006358:Hot Temperature; D006801:Humans; D006860:Hydrogen Bonding; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007231:Infant, Newborn; D007700:Kinetics; D049449:Luminescence; D008297:Male; D008958:Models, Molecular; D009038:Motion; D020125:Mutation, Missense; D010649:Phenylalanine / Q000097:blood / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000032:analysis / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D010661:Phenylketonurias / Q000201:enzymology* / Q000235:genetics*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D001708:Biopterins; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase; D019286:Endopeptidase K; C003402:sapropterin