Thermal equilibrium of high- and low-spin forms of cytochrome P450BM-3: Repositioning of the substrate?
Thermal equilibrium of high- and low-spin forms of cytochrome P450BM-3: Repositioning of the substrate?
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DOI：
10.1021/ja0524604
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发表时间：
2005-10-05
影响因子：
15
通讯作者：
McDermott, AE
中科院分区：
文献类型：
--
作者：
Jovanovic, T;Farid, R;McDermott, AE
我们证明，与N -棕榈酰甘氨酸复合的细胞色素P450 BM - 3在室温（此时可观察到最佳活性）和低温（已进行X射线衍射表征的温度）之间会发生自旋态变化。基于对全长蛋白质的核磁共振测量，这种自旋态变化可能伴随着酶口袋中的一般结构重排。底物在所有温度下都保持结合状态。我们提出，随着温度降低，底物可能从直接位于血红素上方的位置（从而取代配位水）“滑动”到更远的位置（从而恢复配位水）。这一假设是通过对蛋白质 - 配体复合物进行计算建模来评估的，使用了一种新的诱导契合方法。因此，我们生成了一种配体与血红素紧密接触的结构，其能量与实验结构相似。通过理论与实验的结合，我们对该酶的配体如何定位以进行化学反应提出了一个具体的假设。
We demonstrate that cytochrome P450 BM-3 in complex with N-palmitoylglycine undergoes a spin state change between room temperature, where optimal activity is seen, and low temperatures, where X-ray diffraction characterization has been carried out. On the basis of NMR measurements of the full-length protein, this spin state change is likely to be accompanied by a general structural rearrangement in the enzyme pocket. The substrate remains bound at all temperatures. We propose that the substrate may "slide" from a position directly atop the heme (thus displacing the ligating water) to the more distant position (thus restoring the ligating water) as the temperature is lowered. This proposal is evaluated on the basis of computational modeling of the protein-ligand complex, using a novel induced fit methodology. We thereby generate a structure with the ligand in close contact with the heme, similar in energy to the experimental structure. With this combination of theory and experiment we provide a specific proposal of how ligands may be positioned for chemistry for this enzyme.