Open AccessOxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes P ‐450 2C8, 2C9, 2C18 and 2C19
Author(s) -
Jean Pascale,
LopezGarcia Pilar,
Dansette Patrick,
Mansuy Daniel,
Goldstein Joyce L.
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00797.x
Subject(s) - enzyme kinetics , chemistry , hydroxylation , microsome , cytochrome p450 , biochemistry , enzyme , stereochemistry , active site
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 μM, k cat = 1.7 ± 0.2 min −1 ), 30‐fold more potent in terms of k cat /K m than CYP 2C18 ( K m = 150 ± 15 μM, k cat = 1.8 ± 0.2 min −1 and 300‐fold more potent than CYP 2C8 ( K m = 145 ± 15 μM, k cat = 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 t ½ = 10 ± 5 min) during 5‐hydroxylation of tienilic acid.