Structural characterization of the N‐terminal autoregulatory sequence of phenylalanine hydroxylase

Abstract

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.