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J. Biol. Chem., Vol. 280, Issue 13, 12279-12291, April 1, 2005

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Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6^*

Chul-Ho Yun, Keon-Hee Kim, M. Wade Calcutt, and F. Peter Guengerich¶

From the Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea and the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

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 10⁶ M^–1 s^–1), and the k_off 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⁺ ·O₂ 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.

Received for publication, September 24, 2004 , and in revised form, January 18, 2005.

^* This work was supported in part by Korea Research Foundation Grant KRF-2000-015-FS002 and United States Public Health Service Grants R01 CA90426, P30 ES00267, and F32 ES12123. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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