Abstract
Cytochrome P450 2A13-catalyzed α-hydroxylation is a critical step in the activation of the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and (S)-N′-nitrosonornicotine [(S)-NNN]. In the enzyme's active site, a single polar residue, Asn297, can influence substrate binding, orientation, and metabolism. We determined the effects of N297A mutation on enzyme kinetics and specificity for NNK, NNN, and coumarin metabolism. [5-3H]-NNK, [5-3H]-(S)-NNN, [14C]coumarin, and radioflow high-performance liquid chromatography analysis were used to quantify metabolites. Cytochrome P450 (P450) 2A13 N297A catalyzed NNK α-hydroxylation, with a 3-fold preference for methylene versus methyl hydroxylation, similar to wild type. Docking studies using the P450 2A13 crystal structure predicted that when the pyridine ring of NNK cannot hydrogen bond to residue 297 it tilts and orients NNK in positions unfavorable for α-hydroxylation. The N297A mutation resulted in a 5- and 4-fold decrease in catalytic efficiency of NNK and NNN metabolism, respectively, primarily because of increased Km values. The N297A mutation strikingly affected coumarin metabolism. The ratio of coumarin 7-hydroxylation to coumarin 3,4-epoxidation is approximately equal for wild-type enzyme, whereas the ratio was 1:9 for the N297A mutant. Coumarin 3,4-epoxidation was significantly underestimated unless the epoxide was trapped and quantified as its glutathione conjugate. The Km value for this reaction was 4-fold greater for the mutant enzyme; the Vmax value increased nearly 40-fold. The observed shift toward coumarin 3,4-epoxidation is consistent with docking studies. In summary, Asn297 in P450 2A13 is important for orienting NNK and coumarin in the active site, changing this residue to Ala results in altered enzyme kinetics for NNK, NNN, and coumarin.
Footnotes
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This work was supported in part by the National Institutes of Health National Cancer Institute [Grant R01-CA84529]; the National Institutes of Health National Institute on Drug Abuse [Grant F30-DA020968], predoctoral fellowship; and the National Institutes of Health National Cancer Institute [Grant P30-CA77598], mass spectrometry in the Analytical Biochemistry Core.
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J.Z.B. and K.E.S. contributed equally to this work.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.025072.
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ABBREVIATIONS: (S)-NNN, (S)-N′-nitrosonornicotine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; keto alcohol, 4-oxo-4-(3-pyridyl)-1-butanol; keto aldehyde, 4-oxo-4-(3-pyridyl)-1-butanone; lactol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran; P450, cytochrome P450; o-HPA, o-hydroxyphenylacetaldehyde; HPLC, high-performance liquid chromatography; GSH, glutathione; GST, glutathione transferase; LC/MS/MS, liquid chromatography-tandem mass spectrometry; ESI, electrospray ionization.
- Received October 8, 2008.
- Accepted December 10, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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