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Dissecting the molecular basis of a phenylketonuria‐causing mutation in phenylalanine hydroxylase
Author(s) -
Khan Crystal A.,
Meisburger Steve P.,
Ando Nozomi,
Fitzpatrick Paul F.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.528.1
Subject(s) - phenylalanine hydroxylase , phenylalanine , tetrahydrobiopterin , chemistry , allosteric regulation , tyrosine , dimer , phenylketonurias , enzyme , tyrosine hydroxylase , biochemistry , stereochemistry , cofactor , amino acid , organic chemistry
Phenylketonuria (PKU) is a metabolic disorder in which the conversion of excess dietary phenylalanine to tyrosine is disrupted. Phenylalanine hydroxylase (PheH) is responsible for this hydroxylation reaction and uses tetrahydrobiopterin (BH 4 ) and molecular oxygen as cosubstrates. The prevalence of PKU in the U.S. is 1 in 10,000 newborns and was the leading cause of mental retardation prior to the discovery of treatment by a protein‐restricted diet. R68S PheH has been shown to cause a mild form of PKU but the mechanism of action has been controversial. Studies have reported that mutations to Arg68 cause the enzyme to become fully active and lose sensitivity to phenylalanine as an allosteric activator. Arg68 is located in the regulatory domain of PheH, which is responsible for the activation of the enzyme. The current model for activation states that the regulatory domains of two subunits on the tetrameric PheH exist in a monomer‐dimer equilibrium and the dimer is stabilized by binding of phenylalanine. We characterized R68S PheH and found that the R68S PheH is fully active compared to the wild‐type enzyme. Sedimentation velocity experiments showed the isolated regulatory domain of R68S PheH does experience a concentration‐dependent shift from a monomer to a dimer; however, phenylalanine had no effect on this shift. The mutant protein displayed a fluorescence emission spectrum different from that of the wild‐type protein that is unchanged in the presence of phenylalanine. SAXS experiments showed no transitions when the protein was treated with 1 mM phenylalanine. Limited proteolysis experiments showed R68S PheH was more proteolytically sensitive than wild‐type PheH. The data suggest that R68S PheH exhibits constitutively active behavior due to the destabilization of the regulatory domain, which causes the protein to be more proteolytically sensitive. Support or Funding Information NIH F31GM116452 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .