Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability

TY - JOUR

T1 - Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability

AU - Gersting, Søren W

AU - Kemter, Kristina F

AU - Staudigl, Michael

AU - Messing, Dunja D

AU - Danecka, Marta K

AU - Lagler, Florian B

AU - Sommerhoff, Christian P

AU - Roscher, Adelbert A

AU - Muntau, Ania C

PY - 2008/7

Y1 - 2008/7

N2 - 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.

AB - 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.

KW - Administration, Oral

KW - Allosteric Regulation

KW - Amino Acid Metabolism, Inborn Errors

KW - Amino Acid Sequence

KW - Amino Acid Substitution

KW - Binding Sites

KW - Biopterin

KW - Catalytic Domain

KW - Computer Simulation

KW - Dimerization

KW - Endopeptidase K

KW - Enzyme Stability

KW - Female

KW - Hot Temperature

KW - Humans

KW - Hydrogen Bonding

KW - Hydrolysis

KW - Hydrophobic and Hydrophilic Interactions

KW - Infant, Newborn

KW - Kinetics

KW - Luminescence

KW - Male

KW - Models, Molecular

KW - Motion

KW - Mutation, Missense

KW - Phenylalanine

KW - Phenylalanine Hydroxylase

KW - Phenylketonurias

KW - Protein Conformation

KW - Protein Denaturation

KW - Protein Folding

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Protein Subunits

KW - Recombinant Fusion Proteins

KW - Static Electricity

U2 - 10.1016/j.ajhg.2008.05.013

DO - 10.1016/j.ajhg.2008.05.013

M3 - SCORING: Journal article

C2 - 18538294

VL - 83

SP - 5

EP - 17

JO - AM J HUM GENET

JF - AM J HUM GENET

SN - 0002-9297

IS - 1

ER -