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Biochemical characterization of mutant phenylalanine hydroxylase enzymes and correlation with clinical presentation in hyperphenylalaninaemic patients

  • BH4 and PKU
  • Published:
Journal of Inherited Metabolic Disease

Summary

The biochemical properties of mutant phenylalanine hydroxylase (PAH) enzymes and clinical characteristics of hyperphenylalaninaemic patients who bear these mutant enzymes were investigated. Biochemical characterization of mutant PAH enzymes p.D143G, p.R155H, p.L348V, p.R408W and p.P416Q included determination of specific activity, substrate activation, V max, K m for (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4), K d for BH4, and protein stabilization by BH4. Clinical data from 22 patients either homozygous, functionally hemizygous, or compound heterozygous for the mutant enzymes of interest were correlated with biochemical parameters of the mutant enzymes. The p.L348V and p.P416Q enzymes retain significant catalytic activity yet were observed in classic and moderate PKU patients. Biochemical studies demonstrated that BH4 rectified the stability defects in p.L348V and p.P416Q; additionally, patients with these variants responded to BH4 therapy. The p.R155H mutant displayed low PAH activity and decreased apparent affinity for l-Phe yet was observed in mild hyperphenylalaninaemia. The p.R155H mutant does not display kinetic instability, as it is stabilized by BH4 similarly to wild-type PAH; thus the residual activity is available under physiological conditions. The p.R408W enzyme is dysfunctional in nearly all biochemical parameters, as evidenced by disease severity in homozygous and hemizygous patients. Biochemical assessment of mutant PAH proteins, especially parameters involving interaction with BH4 that impact protein folding, appear useful in clinical correlation. As additional patients and mutant proteins are assessed, the utility of this approach will become apparent.

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Abbreviations

BH4 :

(6R)-l-erythro-5,6,7,8-tetrahydrobiopterin

Phe:

phenylalanine

Tyr:

tyrosine

Trp:

tryptophan

CD:

circular dichroism

PAH:

phenylalanine hydroxylase

PKU:

phenylketonuria

MHP:

mild hyperphenylalaninaemia

wt:

wild-type

DTT:

dithiothreitol

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Acknowledgements

We are very grateful to Randi Svebak and Ali Javier Sepulveda for expert technical help. A. L. P. and A. M. are supported by the Research Council of Norway and Helse-Vest. This work was supported by NIH grant 2R44HD075156-02 to S. F. D.

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Authors and Affiliations

  1. Idaho Technology, 390 Wakara Way, Salt Lake City, Utah, 84098, USA

    S. F. Dobrowolski & C. C. Ellingson

  2. Department of Biomedicine, University of Bergen, Bergen, Norway

    A. L. Pey & A. Martinez

  3. University of Southern California, Los Angeles, California, USA

    R. Koch

  4. Children’s Hospital Boston, Boston, Massachusetts, USA

    H. Levy

  5. NGS-Research and Development-GmbH, Medical University of South Carolina, Charleston, South Carolina, USA

    E. W. Naylor

Authors
  1. S. F. Dobrowolski
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  2. A. L. Pey
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  3. R. Koch
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  4. H. Levy
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  5. C. C. Ellingson
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  6. E. W. Naylor
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  7. A. Martinez
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Corresponding author

Correspondence to S. F. Dobrowolski.

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Communicating editor: Nenad Blau

Competing interests: None declared

References to electronic databases: Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/sites/entrez?db = omim. Enzyme Commission: http://www.chem.qmul.ac.uk/iubmb/enzyme/. PAH Mutation database: http://www.pahdb.mcgill.ca/.

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Dobrowolski, S.F., Pey, A.L., Koch, R. et al. Biochemical characterization of mutant phenylalanine hydroxylase enzymes and correlation with clinical presentation in hyperphenylalaninaemic patients. J Inherit Metab Dis 32, 10–21 (2009). https://doi.org/10.1007/s10545-008-0942-6

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  • DOI: https://doi.org/10.1007/s10545-008-0942-6

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