A comparative study of cytochromes P450 2E1 and 2A6: Substrate dynamics, multiple ligand binding, and adduct formation by N-acetyl-m-aminophenol

Cytochrome P450 (CYP) 2E1 and CYP2A6 are two enzymes that are often studied for their involvement in toxic processes (e.g., the activation of tobacco carcinogens and acetaminophen) as well as their roles in drug metabolism (e.g., ethanol and nicotine). These isoforms are good candidates for comparative studies to investigate CYP structure, dynamics, and kinetics. Both isoforms oxidize relatively low molecular weight substrates and also display a rather considerable overlap in substrate selectivity, while simultaneously displaying unique product selectivity, in some instances (e.g., acetaminophen). Using a series of symmetrical, deuterium-labeled substrates (xylene isomers, 2,6-dimethylnaphthalene, and 4,4'-dimethylbiphenyl) the effect of substrate size on substrate dynamics was assessed and compared using intramolecular deuterium isotope effect experiments. As might be expected due to the overlap in substrate selectivity, the profiles of intramolecular isotope effect suppression for CYP2E1 and CYP2A6 were similar with only minor differences. Interestingly, the intramolecular (kH/k D)obs for oxidation of p-xylene-alpha- 2H3 and 2-2H3, 6-dimethylnaphthalene, although statistically significant, were not profoundly different (for either isoform). Therefore, we hypothesized that the intramolecular (kH/k D)obs observed for some xylene isomers are generated from the presence of two ligands. CYP2A6 exhibited cooperative kinetics for m-xylene-alpha-2H3 oxidation, and a concentration-dependent decrease in the m-methylbenzylalcohol/2,4-dimethylphenol product ratio (9.8 +/- 0.1 and 4.8 +/- 0.3 at 2.5 muM and 1000 muM, respectively). Heterotropic effects were observed as well, as incubations containing both 15 muM m-xylene-alpha-2H 3 and 200 muM p-xylene resulted in further reduction of the product ratio. When p-xylene (60 muM) was replaced with deuterium labeled (d6) p-xylene (60 muM) an intermolecular isotope effect on 2,4-dimethylphenol formation from m-xylene-alpha-2H3, (kH/k D)obs = 0.49 was observed, strongly suggesting that p-xylene exerts heterotropic effects by residing in the active site simultaneously with m-xylene. Preliminary evidence is presented that intramolecular isotope effect experiments can be used to provide insight into the molecular mechanisms of P450 allosterism. (Abstract shortened by UMI.)...