Oxidation of tienilic acid by human yeast-expressed cytochromes P-450 2C8, 2C9, 2C18 and 2C19. Evidence that this drug is a mechanism-based inhibitor specific for cytochrome P-450 2C9

Eur J Biochem. 1996 Nov 1;241(3):797-804. doi: 10.1111/j.1432-1033.1996.00797.x.


Oxidation of tienilic acid by human cytochromes P-450 (CYP) 2C9, 2C18, 2C8 and 2C19 was studied using recombinant enzymes expressed in yeast. CYP 2C9 was the best catalyst for 5-hydroxylation of tienilic acid (K(m) = 5 +/- 1 microM, kcat = 1.7 +/- 0.2 min-1), 30-fold more potent in terms of kcat/K(m) than CYP 2C18 (K(m) = 150 +/- 15 microM, kcat = 1.8 +/- 0.2 min-1) and 300-fold more potent than CYP 2C8 (K(m) = 145 +/- 15 microM, kcat = 0.2 +/- 0.1 min-1). CYP 2C19 was unable to catalyze this hydroxylation under our experimental conditions. During this study, a marked effect of the ionic strength on the activities (hydroxylations of tienilic acid and tolbutamide) of these cytochromes P-450 expressed in the yeast strain 334 was observed. The effect was particularly great in the case of CYP 2C18, with a tenfold decrease of activity upon increasing ionic strength from 0.02 to 0.1. Specific-covalent binding of tienilic acid metabolites to cytochrome P-450 (incubations in the presence of 5 mM glutathione) was markedly higher upon tienilic acid oxidation by CYP 2C9 than by CYP 2C18 and CYP 2C8. Mechanism-based inactivation of cytochrome P-450 during tienilic acid oxidation was observed in the case of CYP 2C9 but was not detectable with CYP 2C18 and CYP 2C8. Tienilic acid thus appears to be a mechanism-based inhibitor specific for CYP 2C9 in human liver. Experiments performed with human liver microsomes confirmed that tienilic acid 5-hydroxylase underwent a time-dependent inactivation (apparent t1/2 = 10 +/- 5 min) during 5-hydroxylation of tienilic acid.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aryl Hydrocarbon Hydroxylases*
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Enzyme Inhibitors / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Hydroxylation
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Male
  • Membrane Proteins / metabolism
  • Microsomes / enzymology
  • Microsomes, Liver / enzymology
  • Mixed Function Oxygenases / metabolism
  • Oxidation-Reduction
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Steroid 16-alpha-Hydroxylase*
  • Ticrynafen / metabolism*
  • Ticrynafen / pharmacology
  • Tolbutamide / metabolism


  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Isoenzymes
  • Membrane Proteins
  • Recombinant Proteins
  • Cytochrome P-450 Enzyme System
  • Tolbutamide
  • Mixed Function Oxygenases
  • Aryl Hydrocarbon Hydroxylases
  • Steroid 16-alpha-Hydroxylase
  • Ticrynafen