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.
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.
Stokka
Anne Jorunn
AJ
Section of Biochemistry and Molecular Biology, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway.
Carvalho
Raquel Negrão
RN
Barroso
João Filipe
JF
Flatmark
Torgeir
T
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
D014364:Tryptophan; D010651:Phenylalanine Hydroxylase; D014357:Trypsin