Phosphorylation and Mutations of Ser16 in Human Phenylalanine Hydroxylase

• Published in: The Journal of biological chemistry 2002-10, Vol.277 (43), p.40937-40943
• Main Authors: Miranda, Frederico Faria, Teigen, Knut, Thórólfsson, Matthı́as, Svebak, Randi M., Knappskog, Per M., Flatmark, Torgeir, Martı́nez, Aurora
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112197200

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser 16 by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate l -Phe. So far there is no structural information on the flexible N-terminal tail (residues 1–18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser 16 induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg 13 as well as a repulsion by Glu 280 in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met 1 –Leu 15 ) on phosphorylation is in agreement with the observed conformational change and increased accessibility of the substrate to the active site, as indicated by circular dichroism spectroscopy and the enzyme kinetic data for the full-length phosphorylated and nonphosphorylated human PAH. To further validate the model we have prepared and characterized mutants substituting Ser 16 with a negatively charged residue and found that S16E largely mimics the effects of phosphorylation of human PAH. Both the phosphorylated enzyme and the mutants with acidic side chains instead of Ser 16 revealed an increased resistance toward limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of α-helical structure. In agreement with the modeled structure, the formation of an Arg 13 to Ser 16 phosphate salt bridge and the conformational change of the N-terminal tail also explain the higher stability toward limited tryptic proteolysis of the phosphorylated enzyme. The results obtained with the mutant R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.