Reduced Catalytic Activity of P450 2A6 Mutants with Coumarin: A Computational Investigation.
Reduced Catalytic Activity of P450 2A6 Mutants with Coumarin: A Computational Investigation.
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DOI：
10.1021/ct900018t
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发表时间：
2009-03
影响因子：
5.5
通讯作者：
Weihua Li;H. Ode;T. Hoshino;Hong Liu;Yun Tang;Hualiang Jiang
中科院分区：
文献类型：
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作者：
Weihua Li;H. Ode;T. Hoshino;Hong Liu;Yun Tang;Hualiang Jiang
人细胞色素P450 2A 6是催化香豆素7-羟基化的主要酶，该酶在尼古丁等烟草特有化合物的代谢中也起着重要作用。最近的实验数据表明，P450 2A 6的N297 S和A481 T突变体对香豆素的催化活性分别降低了约4倍和10倍。这两个突变体也有约30倍的香豆素的结合亲和力下降时，相比其野生型。然而，目前，突变如何影响酶活性和/或底物结合仍不清楚。本文采用分子对接和分子动力学模拟相结合的方法对上述问题进行了研究。结果表明，N297 S突变改变了B '-C环和I螺旋之间由水分子介导的氢键网络，从而改变了B'螺旋/B '-C环区域，而A481 T突变通过间接方式触发了其相邻残基包括Phe 209和Phe 280的构象变化，从而影响底物结合。然而，突变并没有显着改变底物的结合方向，因为在活性位点中的唯一极性残基297提供了氢键供体，以指导香豆素的结合。这两种突变都扰乱了活性中心的“Phe簇”的形状，从而削弱了与香豆素的相互作用。计算的结合自由能与实验结合亲和力的相对效力一致。
Human cytochrome P450 2A6 is the major enzyme to catalyze coumarin 7-hydroxylation, and this enzyme also plays an important role in the metabolism of nicotine and other tobacco-specific compounds. Recent experimental data showed that the N297S and A481T mutants of P450 2A6 decreased the catalytic activity toward coumarin by about 4-fold and 10-fold, respectively. These two mutants also had about 30-fold decrease in binding affinity for coumarin when compared to its wild type. At present, however, how the mutations affect the enzymatic activity and/or the substrate binding remains unclear. In this study, a combination of molecular docking and molecular dynamics (MD) simulation was employed to investigate the above question. Our results demonstrated that the N297S mutation altered the hydrogen-bonding network mediated by a water molecule between the B'-C loop and the I helix and thus a shift of the B' helix/B'-C loop region, whereas the A481T mutation triggered the conformational changes of its adjacent residues including Phe209 and Phe280 via an indirect manner to affect the substrate binding. However, the mutations did not significantly alter the substrate binding orientation because the only polar residue 297 in the active site provided the hydrogen-bonding donor to guide the binding of coumarin. Both mutations perturbed the shape of "Phe-cluster" in the active site and thus weakened the interactions with coumarin. The calculated binding free energies were in agreement with the relative potency of the experimental binding affinities.