Identification of the allosteric site for phenylalanine in rat phenylalanine hydroxylase

Shengnan Zhang, Paul F. Fitzpatrick

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEsbetween unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, andH64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH.

Original languageEnglish (US)
Pages (from-to)7418-7425
Number of pages8
JournalJournal of Biological Chemistry
Issue number14
StatePublished - Apr 1 2016

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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