Phosphorylation and Mutations of Ser in Human Phenylalanine Hydroxylase

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser 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 induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg as well as a repulsion by Glu in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met– Leu) 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 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 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 Arg to Ser 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. Phenylalanine hydroxylase (PAH; EC 1.14.16.1, phenylalanine 4-monooxygenase) belongs to the family of aromatic amino acid hydroxylases. PAH catalyzes the hydroxylation of L-Phe to L-Tyr, the rate-limiting step in the catabolism of L-Phe (1, 2), and it requires a nonheme iron, molecular oxygen, and a pterin cofactor for catalysis (3). Genetic defects in the human enzyme (hPAH) cause phenylketonuria, with a broad range of metabolic and clinical phenotypes (4) as well as enzymatic phenotypes (5, 6). hPAH is a tetrameric/dimeric enzyme, and crystal structure analyses (7–9) have shown that each of the chains folds into three domains, i.e. an N-terminal regulatory domain (residues 1–110) that includes the single phosphorylation site Ser, a middle catalytic domain, and a C-terminal oligomerization domain. Mammalian PAH is activated severalfold by preincubation with its substrate L-Phe, which represents the most important mechanism for its regulation in hepatocytes (1). Phosphorylation of PAH at Ser by cAMP-dependent protein kinase (PKA) represents an additional post-transcriptional regulation of the enzyme (10–14). The two mechanisms of activation are interdependent, i.e. L-Phe enhances its rate of phosphorylation by PKA, and the phosphorylated enzyme requires a lower concentration of substrate for its activation (15, 16). It appears that these two mechanisms act synergistically also in vivo and that L-Phe promotes the phosphorylation and activation of PAH in rat liver (13, 17). However, the molecular mechanism of this interdependence is not well understood (18, 19) and has not been explained by the crystal structure analyses of the phosphorylated (at Ser) C24-truncated dimeric form of rat PAH (rPAH) (9). Phosphorylation of the human enzyme results in a mobility shift on SDS-PAGE (20) that is also observed when hPAH is expressed in Escherichia coli (16, 21) and in the in vitro transcription-translation system (5, 22). The enzyme expressed in the latter system is recovered as a double band on SDS-PAGE, corresponding to the phosphorylated ( 51 kDa) and nonphosphorylated ( 50 kDa) forms. Furthermore, we have previously shown by Fourier transform infrared spectroscopy that phosphorylation of the isolated recombinant Nterminal regulatory domain (residues 2–110) results in an apparent increase in the content of -helical structure (23). In the present work we have extended these studies, including molecular modeling by the anchor grow method using the program DOCK 4.0 (24), and the possible conformations of the 18-residue N-terminal tail have been estimated for the nonphospho* This work was supported by the Fundação para a Ciência e a Tecnologia, Portugal, The Research Council of Norway and L. Meltzers Høyskolefond. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains QuickTime videos. ¶ To whom correspondence should be addressed. Tel.: 47-55586427; Fax: 47-55586400; E-mail: [email protected]. 1 The abbreviations used are: PAH, phenylalanine hydroxylase; BH4, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin; hPAH, human phenylalanine hydroxylase; PKA, cyclic AMP-dependent protein kinase; rPAH, rat phenylalanine hydroxylase; wt-hPAH, wild-type hPAH. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 43, Issue of October 25, pp. 40937–40943, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.