Cytochrome P450 2A13-catalyzed alpha-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-(3)H]-NNK, [5-(3)H]-(S)-NNN, [(14)C]coumarin, and radioflow high-performance liquid chromatography analysis were used to quantify metabolites. Cytochrome P450 (P450) 2A13 N297A catalyzed NNK alpha-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 alpha-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 K(m) 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 K(m) value for this reaction was 4-fold greater for the mutant enzyme; the V(max) 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.