Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase.
Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.
Horne
James
J
Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, 3065, Australia.
Jennings
Ian G
IG
Teh
Trazel
T
Gooley
Paul R
PR
Kobe
Bostjan
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005786:Gene Expression Regulation; D007313:Insecta / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010455:Peptides / Q000138:chemical synthesis; D010649:Phenylalanine / Q000378:metabolism / Q000494:pharmacology; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D051381:Rats; D013057:Spectrum Analysis
D010455:Peptides; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases