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J. Biol. Chem., Vol. 277, Issue 43, 40937-40943, October 25, 2002

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Phosphorylation and Mutations of Ser¹⁶ in Human Phenylalanine Hydroxylase
KINETIC AND STRUCTURAL EFFECTS^*,

Frederico Faria Miranda, Knut Teigen, Matthías Thórólfsson, Randi M. Svebak, Per M. Knappskog^§, Torgeir Flatmark, and Aurora Martínez^¶

From the  Department of Biochemistry and Molecular Biology, University of Bergen, Årstadveien 19, 5009-Bergen, Norway and the ^§ Department of Medical Genetics, Haukeland Hospital, University of Bergen, N-5009 Bergen, Norway

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser¹⁶ 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 Ser¹⁶ induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg¹³ as well as a repulsion by Glu²⁸⁰ in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met¹-Leu¹⁵) 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 Ser¹⁶ 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 Ser¹⁶ revealed an increased resistance toward limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of -helical structure. In agreement with the modeled structure, the formation of an Arg¹³ to Ser¹⁶ 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.

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