Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo[a]pyrene-7R-trans-7,8-dihydrodiol

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Abstract
Genetic differences that underlie inter-individual variation in the metabolism of common carcinogens are important potential sources of cancer susceptibility. Cytochrome P450 1B1 (CYP1B1), a central enzyme in the activation of the ubiquitous environmental carcinogen benzo[ a ]pyrene (B[ a ]P), has several genetic variants. This study investigated six rare mutations and four common polymorphisms for their effects on B[ a ]P metabolism. Five missense mutations associated with congenital glaucoma (Gly61Glu, Gly365Trp, Asp374Asn, Pro437Leu and Arg469Tryp) dramatically decreased the capacity of CYP1B1 to convert (−)benzo[ a ]pyrene-7R- trans -7,8-dihyrodiol (B[ a ]P-7,8-diol) to (±)benzo[ a ]pyrene-r-7,t-8-dihydrodiol-9,10-epoxides. These five mutations resulted in enzymes with 3–12% of normal activity when assayed in vitro using an Saccharomyces cerevisiae microsomal expression system. A 10 bp deletion mutation produced no detectable protein or activity. In contrast, proteins containing all possible combinations of four common single nucleotide polymorphisms (Arg48Gly, Ala199Ser, Val432Leu, Asn453Ser) had modest effects on B[ a ]P-7,8-diol metabolism. Michaelis–Menten analysis suggested that two alleles, Arg48, Ala119, Val432, Ser453 (RAVS) and Arg48, Ala119, Leu432, Ser453 (RALS), have KM values 2-fold lower than Arg48, Ala119, Val432, Ser453 (RAVN): 1.4 ± 0.3 and 1.3 ± 0.4 µM, respectively, compared with 2.8 ± 0.8 µM ( P < 0.05). However, these differences could not be confirmed with direct measurements of rate at low substrate concentration. There were no significant differences for either of two other kinetic parameters, kcat or kcat / KM . Allele frequency analysis in three populations reveals the Ser453 variant is rare among those of Asian (a ]P-7,8-diol; however, our results do not identify any major differences in this metabolism due to four common single amino acid polymorphisms.

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