Measurement of Phenylalanine Monooxygenase (PAH) Activities

Glyn B. Steventon1, Stephen C. Mitchell2

1 King's College London, London, United Kingdom, 2 Imperial College London, London, United Kingdom
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.29
DOI:  10.1002/0471140856.tx0429s41
Online Posting Date:  August, 2009
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Abstract

Mammalian phenylalanine monooxygenase (phenylalaninase, phenylalanine hydroxylase, PAH; EC 1.14.16.1) is a member of the large aromatic amino acid hydrolase cohort of enzymes that include tyrosine monooxygenase and tryptophane monooxygenase. PAH is a non‐heme‐iron‐dependent protein that normally catalyzes the C‐oxidation of phenylalanine (Phe) to tyrosine (Tyr) in the presence of BH4, utilizing molecular dioxygen as an additional substrate. However, over recent years, the presumed narrow substrate specificity of PAH has been questioned and catalytic activity towards alternative xenobiotic substrates (both environmental and drugs) has been reported. Like the cytochrome P450 system, PAH is able to oxidize both aliphatic and aromatic carbon centers in addition to undertaking the S‐oxidation of aliphatic thioethers (including the two mucoactive drugs S‐carboxymethyl‐L‐cysteine and S‐methyl‐L‐cysteine). Curr. Protoc. Toxicol. 41:4.29.1‐4.29.11. © 2009 by John Wiley & Sons, Inc.

Keywords: phenylalanine monooxygenase; S‐oxidation; S‐carboxymethyl‐L‐cysteine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring PAH Activity HPLC with Fluorescence Detection
  • Alternate Protocol 1: Measuring PAH Activity Using HPLC with Precolumn Derivitization and Fluorescence Detection
  • Basic Protocol 2: Photometric Determination of PAH Activity
  • Support Protocol 1: Preparation of Cytosol from Cells
  • Support Protocol 2: Protein Determination
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring PAH Activity HPLC with Fluorescence Detection

  Materials
  • 50 mM PIPES buffer (pH 6.8) containing 1 mg/ml bovine catalase (see recipe)
  • 5 µg/ml cDNA expressed PAH or 100 µg/ml hepatic cytosol (pooled or individual human, rat, mouse, or rabbit cytosols were purchased from BD BioSciences)
  • Substrate (Phe; see recipe)
  • 500 µM BH 4 in 30 mM DTT (see recipe)
  • 20% (v/v) TCA (see recipe)
  • 50 mM PIPES buffer (pH 6.8) containing 5 mM lysophosphatidylcholine and 1 mg/ml bovine catalase
  • 1.5‐ml plastic microcentrifuge tubes
  • 37°C water bath
  • HPLC system consisting of:
    • AS3000 autosampler (Thermo Separation)
    • P4000 quaternary gradient pump (Thermo Separation)
    • FL3000 fluorescence detector (Thermo Separation)
    • Data analysis by ChromQuest chromatography data system (Thermo Separation)
    • Spherisorb C 18 10‐µm column (250 × 4.6–mm i.d.; Phenomenex)
  • 2.0‐ml autosampler vial

Alternate Protocol 1: Measuring PAH Activity Using HPLC with Precolumn Derivitization and Fluorescence Detection

  Materials
  • 50 mM PIPES buffer (pH 6.8) containing 1 mg/ml bovine catalase (see recipe)
  • Substrate (S‐carboxymethyl‐L‐cysteine, SCMC; 25 mM, Sigma‐Aldrich)
  • 5 µg/ml cDNA‐expressed PAH or 100 µg/ml hepatic cytosol (pooled or individual human, rat, mouse, or rabbit cytosols were purchased from BD BioSciences)
  • 500 µM BH 4 in 30 mM DTT (see recipe)
  • 20% (v/v) TCA (see recipe)
  • 10 mM cysteic acid
  • 50 mM PIPES buffer (pH 6.8) containing 5 mM lysophosphatidylcholine and 1 mg/ml bovine catalase
  • 1.5‐ml plastic microcentrifuge tubes
  • 37°C water bath
  • HPLC system consisting of:
    • AS3000 autosampler (Thermo Separation)
    • P4000 quaternary gradient pump (Thermo Separation)
    • FL3000 fluorescence detector (Thermo Separation)
    • Data analysis by ChromQuest chromatography data system (Thermo Separation)
    • Hypersil‐OPA amino acid C 18 5‐µm column (30 × 2.1–mm id, Thermo Separation)

Basic Protocol 2: Photometric Determination of PAH Activity

  Materials
  • 50 mM PIPES buffer (pH 6.8) containing 1 mg/ml bovine catalase (see recipe)
  • 5 µg/ml cDNA‐expressed PAH or 100 µg/ml hepatic cytosol (pooled or individual human, rat, mouse, or rabbit cytosols were purchased from BD BioSciences)
  • 2 mM NADH (see recipe)
  • 100 U/ml dihydropteridine reductase
  • Substrate (40 mM Phe and 25 mM SCMC; see recipe)
  • 500 mM BH 4 in 5 mM HCl (see recipe)
  • 50 mM PIPES buffer (pH 6.8) containing 5 mM lysophosphatidylcholine and 1 mg/ml bovine catalase
  • Dihydropteridine reductase (100 U/ml)
  • 1.0‐ml semi‐micro cuvettes, 1‐cm path length
  • UV/VIS spectrophotometer with computer and temperature‐controlled cuvette holder

Support Protocol 1: Preparation of Cytosol from Cells

  Materials
  • Cells (e.g., hepatocytes or HepG2 cells)
  • Phosphate‐buffered saline (PBS; see recipe or appendix 2A)
  • 50 mM PIPES buffer (pH 6.8)
  • 5‐ml round‐bottomed tubes
  • Sonicator with narrow probe
  • Potter‐Elvehjem homogenizer with motorized Teflon pestle
  • Microscope
  • 2‐ml centrifuge tubes
  • 5‐ml ultracentrifuge tubes
  • Ultracentrifuge
NOTE: All procedures are performed at 4°C.

Support Protocol 2: Protein Determination

  Materials
  • BSA standards and unknown sample
  • Coomassie Plus Reagent (Sigma‐Aldrich)
  • 3‐ml test tubes
  • Spectrophotometer
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Figures

Videos

Literature Cited

   Boonyapiwat, B., Panagopoulos, P., Jones, H., Mitchell, S.C., Forbes, B., and Steventon, G.B. 2005. Phenylalanine 4‐monooxygenase and the S‐oxidation of S‐carboxymethyl‐L‐cysteine in HepG2 cells. Drug Metab. Drug Inter. 21:1‐18.
   Boonyapiwat, B., Forbes, B., Mitchell, S.C., and Steventon, G.B. 2008. Phenylalanine 4‐monooxygenase and the S‐oxidation of S‐carboxymethyl‐L‐cysteine by human cytosolic fractions. Drug Metab. Drug Inter. 23:261‐282.
   Boonyapiwat, B., Panaretou, B., Forbes, B., Mitchell, S.C., and Steventon, G.B. 2009. Human phenylalanine monooxygenase and thioether metabolism. J. Pharm. Pharmacol. 61:63‐67.
   Fitzpatrick, P.F. 1999. Tetrahydropterin‐dependent amino acid hydroxylases. Ann. Rev. Biochem. 68:355‐381.
   Goreish, A.H., Bednar, S., Jones, H., Mitchell, S.C., and Steventon, G.B. 2004. Phenylalanine 4‐monooxygenase and the S‐oxidation of S‐carboxymethyl‐L‐cysteine, Drug Metab. Drug Inter. 20:159‐174.
   Hufton, S.E., Jennings, I.G., and Cotton, R.G. 1995. Structure and function of the aromatic amino acid hydroxylases. Biochem. J. 311:353‐366.
   Kaufman, S. and Mason, K. 1982. Specificity of amino acids as activators and substrates for phenylalanine hydroxylase. J. Biol. Chem. 257:14667‐14678.
   Mitchell, S.C. and Waring, R.H. 1989. The deficiency of sulfoxidation of S‐carboxymethyl‐L‐cysteine. Pharmacol. Therap. 43:237‐249.
   Mitchell, S.C., Waring, R.H., Haley, C.S., Idle, J.R., and Smith, R.L. 1984. Genetic aspects of the polymodally distributed sulphoxidation of S‐carboxymethyl‐L‐cysteine in man. Brit. J. Clin. Pharmacol. 18:507‐521.
   PAHdb. 2007. McGill University, Canada, PAHdb Phenylalanine hydroxylase Locus Knowledgebase. http://www.pahdb.mcgill.ca.
   Patel, G.L., Illoudi, C., Boonyapiwat, B., Barlow, D.J., Forbes, B., Mitchell, S.C., and Steventon, G.B. 2008. Enzyme kinetic and molecular modeling studies of sulfur containing substrates of phenylalanine 4‐monooxygenase. J. Enzy. Inhib. Med. Chem. 23:958‐963.
   Steventon, G.B. 1999. Diurnal variation in the metabolism of S‐carboxymethyl‐L‐cysteine in man. Drug Metab. Disp. 27:1092‐1097.
   Steventon, G.B. and Mitchell, S.C. 2006a. The sulphoxidation of S‐carboxymethyl‐L‐cysteine in COPD. Eur. Resp. J. 27:865‐866.
   Steventon, G.B. and Mitchell, S.C. 2006b. Efficacy of S‐carboxymethyl‐L‐cysteine for otitis media with effusion. ENT 85:296‐297.
   Steventon, G.B., Sturman, S., Waring, R.H., and Williams, A.C. 2001. A review of xenobiotic metabolism enzymes in Parkinson's and motor neurone disease. Drug Metab. Drug Inter. 18:79‐98.
   Thorolfsson, M., Teigen, K., and Martinez, A. 2003. Activation of phenylalanine hydroxylase: Effect of substitutions at Arg68 and Cys237. Biochemistry 42:3419‐3428.
Key References
   Boonyapiwat et al., 2005. See above.
  Key reference with regard HepG2 cell cytosol PAH. A modification of these methods is described in and .
   Boonyapiwat et al., 2008. See above.
  Key reference with regard to cDNA expressed PAH. A modification of these methods is described in and .
   Boonyapiwat et al., 2009. See above.
  Key reference with regard to human hepatic cytosol PAH. A modification of these methods is described in and .
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