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J. Biol. Chem., Vol. 280, Issue 20, 19496-19506, May 20, 2005

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Cytochrome P450 3A4-catalyzed Testosterone 6-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting Steps^*

Joel A. Krauser and F. Peter Guengerich

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Testosterone 6-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-²H₅; (ii) 6,6-²H₂; (iii) 6-²H; (iv) 6-²H; and (v) 6-³H testosterone. Only the 6-hydrogen was removed by P450 3A4 and not the 6, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the face. Analysis of the rates of hydroxylation of 6-¹H-, 6-²H-, and 6-³H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect (^Dk) of 15. The deuterium isotope effects on k_cat and k_cat/K_m in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6-²H substitution. The high ^Dk value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high ^Dk in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high ^Dk value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.

Received for publication, February 18, 2005 , and in revised form, March 15, 2005.

^* This work was supported in part by U. S. Public Health Service Grants R01 CA90426, P30 ES00267, and T32 ES07028. 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 ¹³C NMR spectrum of 6,6-d₂-testosterone, HPLC/UV, and LC/MS traces of products of testosterone oxidation by a manganoporphyrin complex/PhI = O system, Hill plots (log₁₀ [v/(k_cat - v)] versus log₁₀ [S]) for testosterone 6-hydroxylation shown in Supplemental Figs. 1S-3S.

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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