Harrelson John P, Atkins William M, Nelson Sidney D
School of Pharmacy, Pacific University, HPC-Ste 451, Hillsboro, Oregon 97123, USA.
Biochemistry. 2008 Mar 4;47(9):2978-88. doi: 10.1021/bi702020y. Epub 2008 Feb 5.
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.
配体动力学对细胞色素P450（CYP）变构作用的贡献尚未得到详细研究。基于先前的一项研究，我们推测CYP2A6和CYP2E1可容纳多个二甲苯配体。因此，对于某些二甲苯异构体观察到的分子内（kH/kD）obs值预计将取决于配体浓度，并受到[CYP.二甲苯]和[CYP.二甲苯.二甲苯]等的影响。为了探索这种可能性以及动力学同位素效应在表征变构CYP行为中的作用，我们测定了CYP2E1和CYP2A6氧化间二甲苯-α-（2）H3和对二甲苯-α-（2）H3的稳态动力学、产物比率以及（kH/kD）obs值。有证据表明CYP2A6可容纳多个配体，并且分子内同位素效应实验能够深入了解多配体结合的机制。CYP2A6对间二甲苯-α-（2）H3氧化表现出协同动力学，并且间甲基苄醇与2,4-二甲基苯酚的产物比率呈现浓度依赖性降低（在2.5 microM和1 mM时分别为9.8±0.1和4.8±0.3）。还观察到了异源效应，因为同时含有15 microM间二甲苯-α-（2）H3和200 microM对二甲苯的孵育导致产物比率进一步降低（2.4±0.2）。当用氘标记的d6-对二甲苯（60 microM）替代对二甲苯（60 microM）时，观察到对2,4-二甲基苯酚形成的分子间竞争性反向同位素效应[（kH/kD）obs = 0.49]，这表明对二甲苯通过与间二甲苯同时存在于活性位点而发挥异源效应。数据表明间二甲苯的重排速率存在浓度依赖性降低，因为在代谢转换水平增加的情况下未观察到（kH/kD）obs增加。也就是说，活性位点中第二个二甲苯分子的容纳导致底物动力学降低。
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