title: Activation of phenylalanine hydroxylase: effect of substitutions at Arg68 and Cys237.
abstract: Phenylalanine hydroxylase (PAH) is a multidomain tetrameric enzyme that displays positive cooperative substrate binding. This cooperative response is believed to be of physiological significance as a mechanism that controls L-Phe homeostasis in blood. The substrate induces an activating conformational change in the enzyme affecting the secondary, tertiary, and quaternary structures. Chemical modification and substitution with a negatively charged residue of Cys237 in human PAH (hPAH) also result in activation of the enzyme. As seen in the modeled structure of full-length hPAH, Cys237 is located in the catalytic domain close to residues in the oligomerization and regulatory domains of an adjacent subunit in the dimer, notably to Arg68. This residue is located in a prominent loop (68-75), which also has contacts with the dimerization motif from the same subunit. To investigate further the involvement of Cys237 and Arg68 in the activation of the enzyme, we have prepared mutants of hPAH at these positions, with substitutions of different charge and size. The mutations C237D, R68A, and C237A cause an increase of the basal activity and affinity for L-Phe, while the mutation C237R results in reduced affinity for the substrate and elimination of the positive cooperativity. The conformational changes induced by the mutations were studied by far-UV circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations. All together, our results indicate that the activating mutations induce a series of conformational changes including both the displacement of the inhibitory N-terminal sequence (residues 19-33) that covers the active site and the domain movements around the hinge region Arg111-Thr117, in addition to the rearrangement of the loop 68-75. The same conformational changes appear to be involved in the activation of PAH induced by L-Phe.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Bergen, Arstadveien 19, 5009-Bergen, Norway.
authors.forename: Matthías
authors.initials: M
authors.lastname: Thórólfsson
authors.forename: Knut
authors.initials: K
authors.lastname: Teigen
authors.forename: Aurora
authors.initials: A
authors.lastname: Martínez
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D001120:Arginine; D010651:Phenylalanine Hydroxylase; D003545:Cysteine