TY - JOUR
T1 - Identification of the allosteric site for phenylalanine in rat phenylalanine hydroxylase
AU - Zhang, Shengnan
AU - Fitzpatrick, Paul F.
N1 - Funding Information:
This work was supported by National Institutes of Health Grant GM047291 and The Welch Foundation Grant AQ-1245. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2016/4/1
Y1 - 2016/4/1
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M115.709998
DO - 10.1074/jbc.M115.709998
M3 - Article
C2 - 26823465
AN - SCOPUS:84962829766
SN - 0021-9258
VL - 291
SP - 7418
EP - 7425
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -