Effector-mediated alteration of substrate orientation in cytochrorne P4502C9
Effector-mediated alteration of substrate orientation in cytochrorne P4502C9
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DOI：
10.1021/bi036158o
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发表时间：
2004-06-08
期刊：
影响因子：
2.9
通讯作者：
Tracy, TS
中科院分区：
文献类型：
--
作者：
Hummel, MA;Gannett, PM;Tracy, TS
细胞色素P450 2C 9（CYP 2C 9）介导的氟比洛芬4 '-羟基化被氨苯砜激活，导致氟比洛芬羟基化的K-m降低和V，m增加。先前的光谱结合研究表明，存在氨苯砜时，氟比洛芬与CYP 2C 9的结合增加（K-S降低）。我们假设，这两种化合物是同时在活性位点与氨苯砜的存在下，导致氟比洛芬被定向更接近血红素。T-1弛豫速率测定NMR被用来估计这些化合物的顺磁性血红素铁中心的质子的距离。在存在和不存在100 μ M氨苯砜的情况下，样品含有0.014 μ M CYP 2C 9和145 μ M氟比洛芬。在不存在氨苯砜的情况下，各种氟比洛芬质子与血红素的估计距离为4.2至4.5埃，在存在氨苯砜的情况下为3.2至3.8埃。氟比洛芬的4'质子（代谢位点）在存在氨苯砜的情况下显示出与血红素的距离的最大差异之一，为3.50埃，而在不存在氨苯砜的情况下为4.41埃。氨苯砜质子受影响较小，在不存在氟比洛芬的情况下距离血红素4.40埃，在存在氟比洛芬的情况下距离血红素4.00-4.01埃。还进行了分子模拟研究，以证实氟比洛芬和氨苯砜在CYP 2C 9活性位点的相对取向。在存在氨苯砜的情况下，氟比洛芬的4'质子向CYP 2C 9的血红素铁移动可能在活化中起作用。
Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4'-hydroxylation is activated by the presence of dapsone resulting in reduction of the K-m, for flurbiprofen hydroxylation and an increase in V,m. Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in K-S) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T-1 relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 muM CYP2C9 and 145muM flurbiprofen in the presence and absence of 100muM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 Angstrom in the absence of dapsone and from 3.2 to 3.8 Angstrom in the presence of dapsone. The 4' proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 Angstrom, as compared to the absence of dapsone, 4.41 Angstrom. Dapsone protons were less affected, being 4.40 Angstrom from the heme in the absence of flurbiprofen and 4.00-4.01 Angstrom from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4' proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.