Orientation of Caffeine within the Active Site of Human Cytochrome P450 1A2 Based on NMR Longitudinal (T1) Relaxation Measurements

doi:10.1006/abbi.2000.2073

Archives of Biochemistry and Biophysics

Volume 384, Issue 1, 1 December 2000, Pages 47-58

https://doi.org/10.1006/abbi.2000.2073 Get rights and content

Abstract

Longitudinal (T₁) relaxation studies were performed in order to examine the interaction of caffeine with the heme of human P450 1A2. Addition of caffeine to this P450 resulted in a small, incomplete conversion of the heme from high spin to low spin, as shown by changes in the optical spectrum. Determination of a relatively large dissociation constant (K_S = 2.6 mM) as well as the relative instability of the P450 after 2 h at room temperature necessitated the performance of these experiments at high concentrations (25 mM) of caffeine. The relaxation measurements on the three sets of methyl hydrogens led to the determination of the corresponding distances between the iron and the methyl groups on the bound caffeine as well as the position and orientation of caffeine within the active site of P450 1A2. The three methyl groups were found to be nearly equidistant from the iron (≥4.79–4.89 Å), with slight preference for the N-3 position, and thus, the average position of caffeine was parallel to the heme. In vitro incubations with P450 1A2 and 5 mM caffeine led primarily to paraxanthine formation (N-3 demethylation), as expected. However, with 25 mM substrate, the overall extent of oxidation was doubled and there was more equivalent oxidation at each of the four potential sites on caffeine. This latter observation was consistent with the lack of selective positioning of the N-3 methyl group of caffeine relative to the heme.

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Citation Excerpt :
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¹: Current address: Department of Pharmacology, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109.

²: To whom correspondence should be addressed. Fax: (206) 685-3252, E-mail: [email protected].

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Regular ArticleOrientation of Caffeine within the Active Site of Human Cytochrome P450 1A2 Based on NMR Longitudinal (T1) Relaxation Measurements

Abstract

Biochem. Pharmacol.

Mol. Cell

Biochemistry

J. Biol. Chem.

Pharmacol. Res.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr. B

FEBS Lett.

Methods Enzymol.

J. Biol. Chem.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

Science

Xenobiotica

Chem. Res. Toxicol.

Drug Metab. Rev.

Xenobiotica

Regular Article
Orientation of Caffeine within the Active Site of Human Cytochrome P450 1A2 Based on NMR Longitudinal (T₁) Relaxation Measurements