Kinetic analysis of oxidation of coumarins by human cytochrome P450 2A6.
Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b5 (b5). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k(on) approximately 10(6) m(-1) s(-1)), and the k(off) rates range from 5.7 to 36 s(-1) (at 23 degrees C). Reduction of ferric P450 2A6 is rapid (7.5 s(-1)) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O2 is rapid (k > or = 10(6) m(-1) s(-1)), and the putative Fe2+.O2 complex decayed at a rate of approximately 0.3 s(-1) at 23 degrees C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b5. Kinetic analyses showed that approximately 1/3 of the reduced b5 was rapidly oxidized in the presence of the Fe2+.O2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6-10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s.
Yun
Chul-Ho
CH
Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea.
Kim
Keon-Hee
KH
Calcutt
M Wade
MW
Guengerich
F Peter
FP
D015660:7-Alkoxycoumarin O-Dealkylase / Q000737:chemistry; D000925:Anticoagulants / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology*; D065637:Cytochrome P-450 CYP2A6; D015786:Cytochromes b5 / Q000737:chemistry; D004579:Electron Transport; D004583:Electrons; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D006900:Hydroxylation; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors; D014466:Ultraviolet Rays
D000925:Anticoagulants; D003374:Coumarins; D002244:Carbon; D006859:Hydrogen; D015786:Cytochromes b5; C030123:coumarin; D006899:Mixed Function Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; D010100:Oxygen