Kinetic analysis of oxidation of coumarins by human cytochrome P450 2A6.

Yun CH, Kim KH, Calcutt MW, Guengerich FP
J Biol Chem. 2005 280 (13): 12279-91

PMID: 15665333 · DOI:10.1074/jbc.M411019200

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.

MeSH Terms (29)

7-Alkoxycoumarin O-Dealkylase Anticoagulants Aryl Hydrocarbon Hydroxylases Binding, Competitive Binding Sites Carbon Catalysis Chromatography, High Pressure Liquid Coumarins Cytochrome P-450 CYP2A6 Cytochromes b5 Electrons Electron Transport Humans Hydrogen Hydrogen Bonding Hydroxylation Kinetics Magnetic Resonance Spectroscopy Mass Spectrometry Mixed Function Oxygenases Models, Chemical Oxygen Protein Binding Protein Structure, Tertiary Spectrophotometry Substrate Specificity Time Factors Ultraviolet Rays

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