title: Probing the role of crystallographically defined/predicted hinge-bending regions in the substrate-induced global conformational transition and catalytic activation of human phenylalanine hydroxylase by single-site mutagenesis.
abstract: Phenylalanine hydroxylase (PAH) is generally considered to undergo a large and reversible conformational transition upon l-Phe binding, which is closely linked to the substrate-induced catalytic activation of this hysteretic enzyme. Recently, several crystallographically solvent-exposed hinge-bending regions including residues 31-34, 111-117, 218-226, and 425-429 have been defined/predicted to be involved in the intra-protomer propagation of the substrate-triggered molecular motions generated at the active site. On this basis, single-site mutagenesis of key residues in these regions of the human PAH tetramer was performed in the present study, and their functional impact was measured by steady-state kinetics and the global conformational transition as assessed by surface plasmon resonance and intrinsic tryptophan fluorescence spectroscopy. A strong correlation (r(2) = 0.93-0.96) was observed between the l-Phe-induced global conformational transition and V(max) values for wild-type human PAH and the mutant forms K113P, N223D, N426D, and N32D, in contrast to the substitution T427P, which resulted in a tetrameric form with no kinetic cooperativity. Furthermore, the flexible intra-domain linker region (residues 31-34) seems to be involved in a more local conformational change, and the biochemical/biophysical properties of the G33A/G33V mutant forms support a key function of this residue in the positioning of the autoregulatory sequence (residues 1-30) and thus in the regulation of the solvent and substrate access to the active site. The mutant forms revealed a variably reduced global conformational stability compared with wild-type human PAH, as measured by thermal denaturation and limited proteolysis.
authors.affiliation: Section of Biochemistry and Molecular Biology, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway.
authors.forename: Anne Jorunn
authors.initials: AJ
authors.lastname: Stokka
authors.forename: Raquel Negrão
authors.initials: RN
authors.lastname: Carvalho
authors.forename: João Filipe
authors.initials: JF
authors.lastname: Barroso
authors.forename: Torgeir
authors.initials: T
authors.lastname: Flatmark
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010651:Phenylalanine Hydroxylase / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013696:Temperature; D014357:Trypsin / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
chemical_list: D014364:Tryptophan; D010651:Phenylalanine Hydroxylase; D014357:Trypsin