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. 2003 Feb 1;369(Pt 3):509–518. doi: 10.1042/BJ20021009

Substrate-induced conformational transition in human phenylalanine hydroxylase as studied by surface plasmon resonance analyses: the effect of terminal deletions, substrate analogues and phosphorylation.

Anne J Stokka 1, Torgeir Flatmark 1
PMCID: PMC1223104  PMID: 12379147

Abstract

The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the slow conformational transition (isomerization) which occurs in human phenylalanine hydroxylase (hPAH) on the binding/dissociation of L-phenylalanine (L-Phe). The binding to immobilized tetrameric wt-hPAH resulted in a time-dependent increase in the refractive index (up to approx. 3 min at 25 degrees C) with an end point of approx. 75 RU (resonance units)/(pmol subunit/mm(2)). By contrast, the contribution of binding the substrate (165 Da) to its catalytic core enzyme [DeltaN(1-102)/DeltaC(428-452)-hPAH] was only approx. 2 RU/(pmol subunit/mm(2)). The binding isotherm for tetrameric and dimeric wt-hPAH revealed a [S](0.5)-value of 98+/-7 microM (h =1.0) and 158+/-11 microM, respectively, i.e. for the tetramer it is slightly lower than the value (145+/-5 microM) obtained for the co-operative binding (h =1.6+/-0.4) of L-Phe as measured by the change in intrinsic tryptophan fluorescence. The responses obtained by SPR and intrinsic tryptophan fluorescence are both considered to be related to the slow reversible conformational transition which occurs in the enzyme upon L-Phe binding, i.e. by the transition from a low-activity state ('T-state') to a relaxed high-activity state ('R-state') characteristic of this hysteretic enzyme, however, the two methods reflect different elements of the transition. Studies on the N- and C-terminal truncated forms revealed that the N-terminal regulatory domain (residues 1-117) plus catalytic domain (residues 118-411) were required for the full signal amplitude of the SPR response. Both the on- and off-rates for the conformational transition were biphasic, which is interpreted in terms of a difference in the energy barrier and the rate by which the two domains (catalytic and regulatory) undergo a conformational change. The substrate analogue 3-(2-thienyl)-L-alanine revealed an SPR response comparable with that of L-Phe on binding to wild-type hPAH.

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Selected References

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  1. Abita J. P., Parniak M., Kaufman S. The activation of rat liver phenylalanine hydroxylase by limited proteolysis, lysolecithin, and tocopherol phosphate. Changes in conformation and catalytic properties. J Biol Chem. 1984 Dec 10;259(23):14560–14566. [PubMed] [Google Scholar]
  2. Andersen O. A., Flatmark T., Hough E. High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active Fe(II) form and binary complex with tetrahydrobiopterin. J Mol Biol. 2001 Nov 23;314(2):279–291. doi: 10.1006/jmbi.2001.5061. [DOI] [PubMed] [Google Scholar]
  3. Andersen Ole Andreas, Flatmark Torgeir, Hough Edward. Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine, and its implications for the mechanism of catalysis and substrate activation. J Mol Biol. 2002 Jul 26;320(5):1095–1108. doi: 10.1016/s0022-2836(02)00560-0. [DOI] [PubMed] [Google Scholar]
  4. Bjørgo E., de Carvalho R. M., Flatmark T. A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild-type and Thr427-->Pro mutant human phenylalanine hydroxylase: contribution of the flexible hinge region Asp425-Gln429 to the tetramerization and cooperative substrate binding. Eur J Biochem. 2001 Feb;268(4):997–1005. doi: 10.1046/j.1432-1327.2001.01958.x. [DOI] [PubMed] [Google Scholar]
  5. Boussaad S., Pean J., Tao N. J. High-resolution multiwavelength surface plasmon resonance spectroscopy for probing conformational and electronic changes in redox proteins. Anal Chem. 2000 Jan 1;72(1):222–226. doi: 10.1021/ac990947n. [DOI] [PubMed] [Google Scholar]
  6. Døskeland A. P., Døskeland S. O., Ogreid D., Flatmark T. The effect of ligands of phenylalanine 4-monooxygenase on the cAMP-dependent phosphorylation of the enzyme. J Biol Chem. 1984 Sep 25;259(18):11242–11248. [PubMed] [Google Scholar]
  7. Døskeland A. P., Martinez A., Knappskog P. M., Flatmark T. Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease. Biochem J. 1996 Jan 15;313(Pt 2):409–414. doi: 10.1042/bj3130409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Erlandsen H., Fusetti F., Martinez A., Hough E., Flatmark T., Stevens R. C. Crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria. Nat Struct Biol. 1997 Dec;4(12):995–1000. doi: 10.1038/nsb1297-995. [DOI] [PubMed] [Google Scholar]
  9. Flatmark T., Almås B., Knappskog P. M., Berge S. V., Svebak R. M., Chehin R., Muga A., Martínez A. Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection. Eur J Biochem. 1999 Jun;262(3):840–849. doi: 10.1046/j.1432-1327.1999.00445.x. [DOI] [PubMed] [Google Scholar]
  10. Flatmark T., Stokka A. J., Berge S. V. Use of surface plasmon resonance for real-time measurements of the global conformational transition in human phenylalanine hydroxylase in response to substrate binding and catalytic activation. Anal Biochem. 2001 Jul 15;294(2):95–101. doi: 10.1006/abio.2001.5163. [DOI] [PubMed] [Google Scholar]
  11. Flatmark Torgeir, Stevens Raymond C. Structural Insight into the Aromatic Amino Acid Hydroxylases and Their Disease-Related Mutant Forms. Chem Rev. 1999 Aug 11;99(8):2137–2160. doi: 10.1021/cr980450y. [DOI] [PubMed] [Google Scholar]
  12. Frieden C. Kinetic aspects of regulation of metabolic processes. The hysteretic enzyme concept. J Biol Chem. 1970 Nov 10;245(21):5788–5799. [PubMed] [Google Scholar]
  13. Fusetti F., Erlandsen H., Flatmark T., Stevens R. C. Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria. J Biol Chem. 1998 Jul 3;273(27):16962–16967. doi: 10.1074/jbc.273.27.16962. [DOI] [PubMed] [Google Scholar]
  14. Gestwicki J. E., Hsieh H. V., Pitner J. B. Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance. Anal Chem. 2001 Dec 1;73(23):5732–5737. doi: 10.1021/ac0105888. [DOI] [PubMed] [Google Scholar]
  15. Gjetting T., Petersen M., Guldberg P., Güttler F. Missense mutations in the N-terminal domain of human phenylalanine hydroxylase interfere with binding of regulatory phenylalanine. Am J Hum Genet. 2001 Apr 20;68(6):1353–1360. doi: 10.1086/320604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Iwaki M., Phillips R. S., Kaufman S. Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase. J Biol Chem. 1986 Feb 15;261(5):2051–2056. [PubMed] [Google Scholar]
  17. Jennings I. G., Teh T., Kobe B. Essential role of the N-terminal autoregulatory sequence in the regulation of phenylalanine hydroxylase. FEBS Lett. 2001 Jan 19;488(3):196–200. doi: 10.1016/s0014-5793(00)02426-1. [DOI] [PubMed] [Google Scholar]
  18. Jönsson U., Fägerstam L., Ivarsson B., Johnsson B., Karlsson R., Lundh K., Löfås S., Persson B., Roos H., Rönnberg I. Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques. 1991 Nov;11(5):620–627. [PubMed] [Google Scholar]
  19. Kappock T. Joseph, Caradonna John P. Pterin-Dependent Amino Acid Hydroxylases. Chem Rev. 1996 Nov 7;96(7):2659–2756. doi: 10.1021/cr9402034. [DOI] [PubMed] [Google Scholar]
  20. Kaufman S., Mason K. Specificity of amino acids as activators and substrates for phenylalanine hydroxylase. J Biol Chem. 1982 Dec 25;257(24):14667–14678. [PubMed] [Google Scholar]
  21. Kaufman S. Tyrosine hydroxylase. Adv Enzymol Relat Areas Mol Biol. 1995;70:103–220. doi: 10.1002/9780470123164.ch3. [DOI] [PubMed] [Google Scholar]
  22. Knappskog P. M., Flatmark T., Aarden J. M., Haavik J., Martínez A. Structure/function relationships in human phenylalanine hydroxylase. Effect of terminal deletions on the oligomerization, activation and cooperativity of substrate binding to the enzyme. Eur J Biochem. 1996 Dec 15;242(3):813–821. doi: 10.1111/j.1432-1033.1996.0813r.x. [DOI] [PubMed] [Google Scholar]
  23. Knappskog P. M., Haavik J. Tryptophan fluorescence of human phenylalanine hydroxylase produced in Escherichia coli. Biochemistry. 1995 Sep 19;34(37):11790–11799. doi: 10.1021/bi00037a017. [DOI] [PubMed] [Google Scholar]
  24. Kobe B., Jennings I. G., House C. M., Michell B. J., Goodwill K. E., Santarsiero B. D., Stevens R. C., Cotton R. G., Kemp B. E. Structural basis of autoregulation of phenylalanine hydroxylase. Nat Struct Biol. 1999 May;6(5):442–448. doi: 10.1038/8247. [DOI] [PubMed] [Google Scholar]
  25. Mannen T., Yamaguchi S., Honda J., Sugimoto S., Kitayama A., Nagamune T. Observation of charge state and conformational change in immobilized protein using surface plasmon resonance sensor. Anal Biochem. 2001 Jun 15;293(2):185–193. doi: 10.1006/abio.2001.5141. [DOI] [PubMed] [Google Scholar]
  26. Martinez A., Knappskog P. M., Olafsdottir S., Døskeland A. P., Eiken H. G., Svebak R. M., Bozzini M., Apold J., Flatmark T. Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme. Biochem J. 1995 Mar 1;306(Pt 2):589–597. doi: 10.1042/bj3060589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martínez A., Haavik J., Flatmark T. Cooperative homotropic interaction of L-noradrenaline with the catalytic site of phenylalanine 4-monooxygenase. Eur J Biochem. 1990 Oct 5;193(1):211–219. doi: 10.1111/j.1432-1033.1990.tb19325.x. [DOI] [PubMed] [Google Scholar]
  28. Neet K. E., Ainslie G. R., Jr Hysteretic enzymes. Methods Enzymol. 1980;64:192–226. doi: 10.1016/s0076-6879(80)64010-5. [DOI] [PubMed] [Google Scholar]
  29. Parniak M. A., Kaufman S. Rat liver phenylalanine hydroxylase. Activation by sulfhydryl modification. J Biol Chem. 1981 Jul 10;256(13):6876–6882. [PubMed] [Google Scholar]
  30. Phillips R. S., Iwaki M., Kaufman S. Ligand effects on the limited proteolysis of phenylalanine hydroxylase: evidence for multiple conformational states. Biochem Biophys Res Commun. 1983 Feb 10;110(3):919–925. doi: 10.1016/0006-291x(83)91050-1. [DOI] [PubMed] [Google Scholar]
  31. Phillips R. S., Parniak M. A., Kaufman S. Spectroscopic investigation of ligand interaction with hepatic phenylalanine hydroxylase: evidence for a conformational change associated with activation. Biochemistry. 1984 Aug 14;23(17):3836–3842. doi: 10.1021/bi00312a007. [DOI] [PubMed] [Google Scholar]
  32. Shiman R., Gray D. W., Pater A. A simple purification of phenylalanine hydroxylase by substrate-induced hydrophobic chromatography. J Biol Chem. 1979 Nov 25;254(22):11300–11306. [PubMed] [Google Scholar]
  33. Shiman R., Gray D. W. Substrate activation of phenylalanine hydroxylase. A kinetic characterization. J Biol Chem. 1980 May 25;255(10):4793–4800. [PubMed] [Google Scholar]
  34. Shiman R. Relationship between the substrate activation site and catalytic site of phenylalanine hydroxylase. J Biol Chem. 1980 Nov 10;255(21):10029–10032. [PubMed] [Google Scholar]
  35. Solstad T., Flatmark T. Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids. Effects on catalytic and stability properties. Eur J Biochem. 2000 Oct;267(20):6302–6310. doi: 10.1046/j.1432-1327.2000.01715.x. [DOI] [PubMed] [Google Scholar]
  36. Sota H., Hasegawa Y., Iwakura M. Detection of conformational changes in an immobilized protein using surface plasmon resonance. Anal Chem. 1998 May 15;70(10):2019–2024. doi: 10.1021/ac9713666. [DOI] [PubMed] [Google Scholar]
  37. Teigen K., Frøystein N. A., Martínez A. The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism. J Mol Biol. 1999 Dec 3;294(3):807–823. doi: 10.1006/jmbi.1999.3288. [DOI] [PubMed] [Google Scholar]
  38. Thórólfsson Matthías, Ibarra-Molero Beatriz, Fojan Peter, Petersen Steffen B., Sanchez-Ruiz Jose M., Martínez Aurora. L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study. Biochemistry. 2002 Jun 18;41(24):7573–7585. doi: 10.1021/bi0160720. [DOI] [PubMed] [Google Scholar]
  39. Zako T., Harada K., Mannen T., Yamaguchi S., Kitayama A., Ueda H., Nagamune T. Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance. J Biochem. 2001 Jan;129(1):1–4. doi: 10.1093/oxfordjournals.jbchem.a002818. [DOI] [PubMed] [Google Scholar]

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