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Papers In Press, published online ahead of print August 15, 2002
J. Biol. Chem, 10.1074/jbc.M112197200
Submitted on December 20, 2001
Revised on June 14, 2002
Accepted on August 15, 2002

Phosphorylation and mutations of Ser16 in human phenylalanine hydroxylase. Kinetic and structural effects

Frederico F. Miranda, Knut Teigen, Matthías Thórólfsson, Randi M. Svebak, Per M. Knappskog, Torgeir Flatmark, and Aurora Martínez

Biochemistry and Molecular Biology, University of Bergen, Bergen 5009

Corresponding Author: aurora.martinez{at}ibmb.uib.no

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by PKA 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). In order 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 induces a local conformational change resulting from 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. The 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 non-phosphorylated human PAH (hPAH). In order to further validate the model we have prepared and characterized mutants substituting Ser16 with a negatively charged residue and found that, notably S16E largely mimics the effects of phosphorylation of hPAH. Both the phosphorylated enzyme and the mutants with acidic side-chains instead of Ser16 revealed an increased resistance towards limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of alpha -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 towards limited proteolysis of the phosphorylated enzyme. The results obtained with the mutant forms R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.


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