期刊
BIOCHEMISTRY
卷 47, 期 9, 页码 2978-2988出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi702020y
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资金
- NIEHS NIH HHS [P30ES07033] Funding Source: Medline
- NIGMS NIH HHS [GM 32165] Funding Source: Medline
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 center dot xylene] and [CYP center dot xylene center dot 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-a-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 mu M and 1 mM, respectively). Heterotropic effects were observed as well, as incubations containing both 15 mu M m-xylene-alpha-(2)H(3) and 200 mu M p-xylene resulted in further reduction of the product ratio (2.4 +/- 0.2). When p-xylene (60 mu M) was replaced with deuterium-labeled d(6)-p-xylene (60 mu M), 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.
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