Multiple-Ligand Binding in CYP2A6: Probing Mechanisms of Cytochrome P450 Cooperativity by Assessing Substrate Dynamics

The contribution of ligand dynamics to CYP allosterism has not been considered in detail. On the basis of a previous study, we hypothesized that CYP2A6 and CYP2E1 accommodate multiple xylene ligands. As a result, the intramolecular (k H/k D)obs values observed for some xylene isomers are expected to be dependent on ligand concentration with contributions from [CYP·xylene] and [CYP·xylene·xylene], etc. To explore this possibility and the utility of kinetic isotope effects in characterizing allosteric CYP behavior, steady state kinetics, product ratios, and (k H/k D)obs values for CYP2E1 and CYP2A6 oxidation of m-xylene-α-2H3 and p-xylene-α-2H3 were determined. Evidence is presented that CYP2A6 accommodates multiple ligands and that intramolecular isotope effect experiments can provide insight into the mechanisms of multiple-ligand binding. CYP2A6 exhibited cooperative kinetics for m-xylene-α-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 µM and 1 mM, respectively). Heterotropic effects were observed as well, as incubations containing both 15 µM m-xylene-α-2H3 and 200 µM p-xylene resulted in further reduction of the product ratio (2.4 ± 0.2). When p-xylene (60 µM) was replaced with deuterium-labeled d 6-p-xylene (60 µM), an intermolecular competitive inverse isotope effect on 2,4-dimethylphenol formation [(k H/k D)obs = 0.49] was observed, indicating that p-xylene exerts heterotropic effects by residing in the active site simultaneously with m-xylene. The data indicate that there is a concentration-dependent decrease in the reorientation rate of m-xylene, as no increase in (k H/k D)obs was observed in the presence of an increased level of metabolic switching. That is, the accommodation of a second xylene molecule in the active site leads to a decrease in substrate dynamics.