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

Abstract

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 (kon ∼ 106 M-1 s -1), and the koff rates range from 5.7 to 36 s -1 (at 23 °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 ≥ 106 M-1 s-1), and the putative Fe2+·O 2 complex decayed at a rate of ∼0.3 s-1 at 23 °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 ∼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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.