title: Multiple-ligand binding in CYP2A6: probing mechanisms of cytochrome P450 cooperativity by assessing substrate dynamics.
abstract: 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-alpha- (2)H 3 and p-xylene-alpha- (2)H 3 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-alpha- (2)H 3 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 microM and 1 mM, respectively). Heterotropic effects were observed as well, as incubations containing both 15 microM m-xylene-alpha- (2)H 3 and 200 microM p-xylene resulted in further reduction of the product ratio (2.4 +/- 0.2). When p-xylene (60 microM) was replaced with deuterium-labeled d 6- p-xylene (60 microM), 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.
authors.affiliation: School of Pharmacy, Pacific University, HPC-Ste 451, Hillsboro, Oregon 97123, USA. harrelsonj@pacificu.edu
authors.forename: John P
authors.initials: JP
authors.lastname: Harrelson
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
authors.forename: Sidney D
authors.initials: SD
authors.lastname: Nelson
mesh_terms: D000465:Algorithms; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001665:Binding Sites; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D007697:Kinesics; D007700:Kinetics; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008954:Models, Biological; D008956:Models, Chemical; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011485:Protein Binding; D013379:Substrate Specificity; D014992:Xylenes / Q000737:chemistry / Q000378:metabolism
chemical_list: D008024:Ligands; D014992:Xylenes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6