Kim, KH Isin, EM Yun, CH Kim, DH Guengerich, FP 2024-01-21T03:05:14Z 2024-01-21T03:05:14Z 2021-09-01 2006-05 1742-464X https://pubs.kist.re.kr/handle/201004/135552 7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant. English WILEY ESCHERICHIA-COLI SPECTRAL CHARACTERIZATION HYDROXYLATION 7-ETHOXYCOUMARIN EXPRESSION PURIFICATION SUBSTITUTION 2E1 STABILIZATION DEETHYLATION Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes - Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation Article 10.1111/j.1742-4658.2006.05235.x 1 FEBS JOURNAL, v.273, no.10, pp.2223 - 2231 FEBS JOURNAL 273 10 2223 2231 scie scopus 000237180700012 2-s2.0-33646267951 Biochemistry & Molecular Biology Biochemistry & Molecular Biology Article ESCHERICHIA-COLI SPECTRAL CHARACTERIZATION HYDROXYLATION 7-ETHOXYCOUMARIN EXPRESSION PURIFICATION SUBSTITUTION 2E1 STABILIZATION DEETHYLATION alkoxycoumarins coumarins cytochrome P450 kinetic isotope effects microsomal reactions