Characterization of Purified Human Recombinant Cytochrome P4501A1-Ile⁴⁶² and -Val⁴⁶²: Assessment of a Role for the Rare Allele in Carcinogenesis

Abstract

Human cytochrome P4501A1 (CYP1A1) occurs extrahepatically and is polymorphic, the common form having Ile at position 462 and the rare form having Val. The rare allele has been associated with enhanced susceptibility to lung cancer. To resolve its role in cancer we have constructed CYP1A1-Val⁴⁶² cDNA by site-directed mutagenesis from CYP1A1-Ile⁴⁶², as confirmed by sequencing and allele-specific PCR. Both alleles were expressed in Escherichia coli, and CYP1A1-Ile⁴⁶² and -Val⁴⁶² were purified to electrophoretic homogeneity. The secondary structures of both forms were virtually identical, with high α helix content, as assessed by circular dichroism. The P450s stereoselectively and regioselectively catalyzed the metabolism of (R)- and (S)-warfarin, in reconstituted systems, with very similar profiles. Both P450s produced (R)-6- and 8-hydroxy-warfarin with K_m values of 0.40 ± 0.06 and 0.43 ± 0.05 mm, respectively, and V_max values of 84.0 ± 6.8 and 137.7 ± 8.9 pmol/min/nmol CYP1A1-Val⁴⁶², respectively, 1.0 ± 0.1 and 1.0 ± 0.1 mm, respectively, and 46.7 ± 2.5 and 80.0 ± 4.4 pmol/min/nmol CYP1A1-Ile⁴⁶², respectively. Reconstituted CYP1A1-Val⁴⁶² catalyzed ethoxyresorufin metabolism at a slightly but significantly higher rate than did CYP1A1-Ile⁴⁶²; V_max values were 4.4 ± 0.6 and 3.1 ± 0.3 nmol/min/nmol CYP1A1, respectively. However, with the carcinogen benzo(a)pyrene as substrate, reconstituted CYP1A1-Ile⁴⁶² together with epoxide hydrolase produced 7,8- and 9,10-dihydrodiols at comparable rates than did CYP1A1-Val⁴⁶². Thus, the apparently greater susceptibility of the CYP1A1-Val⁴⁶² genotype to lung cancer is probably not related to greater extents of carcinogen bioactivation.