title: Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase.
abstract: 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 NOEs between 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, and H64N 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.
authors.affiliation: From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229.
authors.forename: Shengnan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229 fitzpatrickp@uthscsa.edu.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010649:Phenylalanine / Q000737:chemistry* / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D051381:Rats
chemical_list: D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase
keywords: allosteric regulation; binding site; enzyme kinetics; nuclear magnetic resonance (NMR); phenylalanine hydroxylase; site-directed mutagenesis; tetrahydrobiopterin (BH4)
references.citation: Mol Biol Med. 1991 Feb;8(1):3-18
references.pmid: 1943687
references.citation: Biochemistry. 1984 Aug 14;23(17):3836-42
references.pmid: 6487579
references.citation: Nat Struct Biol. 1995 Jan;2(1):69-76
references.pmid: 7719856
references.citation: J Biomol NMR. 1995 Nov;6(3):277-93
references.pmid: 8520220
references.citation: Biochem J. 1996 Jan 15;313 ( Pt 2):409-14
references.pmid: 8573072
references.citation: Nat Struct Biol. 1997 Jul;4(7):578-85
references.pmid: 9228951
references.citation: Nat Struct Biol. 1999 May;6(5):442-8
references.pmid: 10331871
references.citation: J Biomol NMR. 2005 May;32(1):71-81
references.pmid: 16041485
references.citation: J Biol Chem. 2006 Nov 10;281(45):33825-9
references.pmid: 16987805
references.citation: Anal Biochem. 2007 Feb 1;361(1):24-30
references.pmid: 17181992
references.citation: J Struct Biol. 2008 Apr;162(1):94-107
references.pmid: 18171624
references.citation: Biochemistry. 2010 Apr 20;49(15):3327-35
references.pmid: 20307070
references.citation: Amino Acids. 2010 Nov;39(5):1463-75
references.pmid: 20480196
references.citation: Arch Biochem Biophys. 2011 Jan 15;505(2):250-5
references.pmid: 20951114
references.citation: Arch Biochem Biophys. 2011 Apr 1;508(1):1-12
references.pmid: 21176768
references.citation: Arch Biochem Biophys. 2012 Mar 15;519(2):194-201
references.pmid: 22005392
references.citation: Arch Biochem Biophys. 2013 Feb 15;530(2):73-82
references.pmid: 23296088
references.citation: IUBMB Life. 2013 Apr;65(4):341-9
references.pmid: 23457044
references.citation: Eur Biophys J. 2013 Sep;42(9):691-708
references.pmid: 23860686
references.citation: J Mol Biol. 2014 Apr 3;426(7):1483-97
references.pmid: 24361276
references.citation: Biochemistry. 2014 Oct 28;53(42):6625-7
references.pmid: 25299136
references.citation: Biochemistry. 2014 Dec 16;53(49):7846-53
references.pmid: 25453233
references.citation: Curr Opin Struct Biol. 2014 Dec;29:102-11
references.pmid: 25543886
references.citation: Biochemistry. 2015 Aug 25;54(33):5167-74
references.pmid: 26252467
references.citation: J Biomol Struct Dyn. 2016;34(3):497-507
references.pmid: 26479306
references.citation: FEBS Lett. 2001 Jan 19;488(3):196-200
references.pmid: 11163771
references.citation: Am J Hum Genet. 2001 Jun;68(6):1353-60
references.pmid: 11326337
references.citation: J Biomol NMR. 2001 May;20(1):71-5
references.pmid: 11430757
references.citation: Biochemistry. 2002 Jun 18;41(24):7573-85
references.pmid: 12056888
references.citation: Biochemistry. 2002 Oct 22;41(42):12569-74
references.pmid: 12379098
references.citation: Biochemistry. 2003 Dec 9;42(48):14083-91
references.pmid: 14640675
references.citation: J Comput Chem. 2004 Oct;25(13):1605-12
references.pmid: 15264254
references.citation: Methods Mol Biol. 2004;278:313-52
references.pmid: 15318002
references.citation: J Biol Chem. 1979 Nov 25;254(22):11300-6
references.pmid: 500646
references.citation: J Biol Chem. 1994 Oct 7;269(40):24647-56
references.pmid: 7929136