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Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations

  1. Heidi Erlandsen * , † , ‡,
  2. Angel L. Pey ‡ , § , ,
  3. Alejandra Gámez *,
  4. Belén Pérez §,
  5. Lourdes R. Desviat §,
  6. Cristina Aguado §,
  7. Richard Koch ∥ , **,
  8. Sankar Surendran ††,
  9. Stephen Tyring ††,
  10. Reuben Matalon ** , ††,
  11. Charles R. Scriver ** , ‡‡,
  12. Magdalena Ugarte §,
  13. Aurora Martínez §§ , ¶¶, and
  14. Raymond C. Stevens * , ** , ¶¶
  1. *Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; §Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas–Universidad Autónoma de Madrid, 28049 Madrid, Spain; ∥Children's Hospital of Los Angeles, Los Angeles, CA 90027; ††Department of Pediatrics and Microbiology, University of Texas Medical Branch, Galveston, TX 77555; ‡‡Montreal Children's Hospital Research Institute, McGill University, 2300 Tupper Street, Montreal, PQ, Canada H3H 1P3; and §§Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway

  1. Communicated by Ernest Beutler, The Scripps Research Institute, La Jolla, CA, September 30, 2004 (received for review July 19, 2004)

Abstract

Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4)]. Several hypotheses have been put forward to explain BH4 responsiveness, but the molecular basis for the corrective effect(s) of BH4 has not been understood. We have investigated the biochemical, kinetic, and structural changes associated with BH4-responsive mutations (F39L, I65T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C). The biochemical and kinetic characterization of the 15 mutants studied points toward a multifactorial basis for the BH4 responsiveness; the mutants show residual activity (>30% of WT) and display various kinetic defects, including increased K m (BH4) and reduced cooperativity of substrate binding, but no decoupling of cofactor (BH4) oxidation. For some, BH4 seems to function through stabilization and protection of the enzyme from inactivation and proteolytic degradation. In the crystal structures of a phenylketonuria mutant, A313T, minor changes were seen when compared with the WT PAH structures, consistent with the mild effects the mutant has upon activity of the enzyme both in vitro and in vivo. Truncations made in the A313T mutant PAH form revealed that the N and C termini of the enzyme influence active site binding. Of fundamental importance is the observation that BH4 appears to increase Phe catabolism if at least one of the two heterozygous mutations has any residual activity remaining.

Footnotes

  • ↵ ¶¶ To whom correspondence may be addressed. E-mail: aurora.martinez{at}ibmb.uib.no or stevens{at}scripps.edu.

  • ↵ † Present address: Department of Biochemistry and Biophysics, Stockholm University, Roslagstullsbacken 15, 114 21, Stockholm, Sweden.

  • ↵ ‡ H.E. and A.L.P. contributed equally to this work.

  • ↵ Present address: Department of Biomedicine, University of Bergen, Jones Lies vei 91, 5009 Bergen, Norway.

  • ↵ ** R.C.S., C.R.S., R.M., and R.K. are advisors for BioMarin (Novato, CA) in the area of BH4 treatment of PKU.

  • Author contributions: H.E., R.K., S.S., S.T., R.M., C.R.S., M.U., A.M., and R.S. designed research; A.L.P., A.G., B.P., L.R.D., C.A., A.M., L.R.D., B.P., and R.S. performed research; A.L.P., A.G., B.P., L.R.D., C.A., A.M., and R.S. contributed new reagents or analytic tools; H.E., A.L.P., A.G., B.P., L.R.D., C.A., R.K., S.S., S.T., R.M., C.R.S., M.U., A.M., and R.S. analyzed data; H.E., A.M., and R.S. wrote the paper.

  • Abbreviations: BH4,(6R)-l-erythro-5,6,7,8-tetrahydrobiopterin; HPA, hyperphenylalaninemia; PAH, phenylalanine hydroxylase; wt-PAH, WT PAH; dt-PAH, double-truncated PAH; PKU, phenylketonuria; RMSD, rms deviation; h, Hill coefficient.

  • Data deposition: The protein structures have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1TDW and 1TG2 for the PKU mutation A313T and the A313T 7,8-BH2-bound form, respectively).

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