Cytochrome P450 2A-Catalyzed Metabolic Activation of Structurally Similar Carcinogenic Nitrosamines:  N‘-Nitrosonornicotine Enantiomers, N-Nitrosopiperidine, and N-Nitrosopyrrolidine

N‘-Nitrosonornicotine (NNN) and N-nitrosopiperidine (NPIP) are potent esophageal and nasal cavity carcinogens in rats and pulmonary carcinogens in mice. N-Nitrosopyrrolidine (NPYR) induces mainly liver tumors in rats and is a weak pulmonary carcinogen in mice. These nitrosamines may be causative agents in human cancer. α-Hydroxylation is believed to be the key activation pathway in their carcinogenesis. P450 2As are important enzymes of nitrosamine α-hydroxylation. Therefore, a structure−activity relationship study of rat P450 2A3, mouse P450 2A4 and 2A5, and human P450 2A6 and 2A13 was undertaken to compare the catalytic activities of these enzymes for α-hydroxylation of (R)-NNN, (S)-NNN, NPIP, and NPYR. Kinetic parameters differed significantly among the P450 2As although their amino acid sequence identities were 83% or greater. For NNN, α-hydroxylation can occur at the 2‘- or 5‘-carbon. P450 2As catalyzed 5‘-hydroxylation of (R)- or (S)-NNN with K m values of 0.74−69 μM. All of the P450 2As except P450 2A6 catalyzed (R)-NNN 2‘-hydroxylation with K m values of 0.73−66 μM. (S)-NNN 2‘-hydroxylation was not observed. Although P450 2A4 and 2A5 differ by only 11 amino acids, they were the least and most efficient catalysts of NNN 5‘-hydroxylation, respectively. The catalytic efficiencies (k cat/K m) for (R)-NNN differed by 170-fold whereas there was a 46-fold difference for (S)-NNN. In general, P450 2As catalyzed (R)- and (S)-NNN 5‘-hydroxylation with significantly lower K m and higher k cat/K m values than NPIP or NPYR α-hydroxylation (p < 0.05). Furthermore, P450 2As were better catalysts of NPIP α-hydroxylation than NPYR. P450 2A4, 2A5, 2A6, and 2A13 exhibited significantly lower K m and higher k cat/K m values for NPIP than NPYR α-hydroxylation (p < 0.05), similar to previous reports with P450 2A3. Taken together, these data indicate that critical P450 2A residues determine the catalytic activities of NNN, NPIP, and NPYR α-hydroxylation.