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Binding and Oxidation of Alkyl 4-Nitrophenyl Ethers by Rabbit Cytochrome P450 1A2:  Evidence for Two Binding Sites

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Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
Cite this: Biochemistry 2001, 40, 24, 7262–7272
Publication Date (Web):May 15, 2001
https://doi.org/10.1021/bi010402z
Copyright © 2001 American Chemical Society

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    Abstract

    Although most cytochrome P450 (P450) reactions demonstrate saturation kinetics that fit to the standard Michaelis−Menten equation, there are important exceptions where sigmoidal or nonhyperbolic behavior is observed and have been fit instead to kinetic models involving two binding sites. To assess these models, we demonstrate the consistency of a two binding site model to interpret both steady-state kinetics and binding events. Rates of 4-nitrophenol and formaldehyde production from the O-demethylation of 1-methoxy-4-nitrobenzene by P450 1A2 isolated from rabbit liver produced biphasic plots, when plotted against substrate concentration. Experiments confirmed the absence of the further oxidation of the products. Recombinant rabbit P450 1A2 yielded the same maximal velocity and more marked biphasicity. Overall, these steady-state data fit well to kinetic models involving two binding sites. Steady-state studies of substrates with bulkier O-ethyl or O-isopropoxy groups indicated decreased affinity for the second site. Based on binding studies, the affinity of P450 1A2 for these substrates increased 200-fold with the larger alkyl groups. To analyze the single binding site model, competition studies were conducted with 1,4-phenyldiisocyanide and the alkyl 4-nitrophenyl ethers. Although the observed dissociation constants and the competing titrant demonstrated a linear dependence, the affinity for the competing titrant depended on the presence of the other titrant, which violates the single binding site model. Alternatively, we applied a two binding site model to these data to obtain dissociation constants for the binary and ternary complexes. The agreement between the dissociation constants for the heterogeneous complexes supports the appropriateness of the two binding site model. This novel finding for P450 1A2 may be more common than originally perceived for P450s.

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     This work was supported in part by U.S. Public Health Service (USPHS) Grants R35 CA44353 and P30 ES00267. G.P.M. was supported in part by USPHS Postdoctoral Fellowship F32 GM19808.

    *

     To whom correspondence should be addressed at the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Medical Research Building I, 23rd and Pierce Ave., Nashville, TN 37232-0146. Tel.:  615-322-2261; Fax:  615-322-3141; E-mail:  [email protected].

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    Figure showing HPLC separation of formaldehyde 2,4-dinitrophenylhydrazone and DYNAFIT scripts used for estimating binding constants for two binding site models (Schemes 3 and 4) (5 pages). This material is available free of charge via the Internet at http://pubs.acs.org.

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