Lin Hsia-Lien, Zhang Haoming, Noon Kathleen R, Hollenberg Paul F
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-5632, USA.
J Pharmacol Exp Ther. 2009 Nov;331(2):392-403. doi: 10.1124/jpet.109.158782. Epub 2009 Aug 21.
The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2-propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a k(inact)/K(I) value of 1343 min(-1)mM(-1) and a partition ratio of 1. The inactivation of WT by BMP exhibited a k(inact)/K(I) value of 33 min(-1)mM(-1) and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the k(inact)/K(I) values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results.
研究了在重组系统中，叔丁基苯乙炔（BPA）和叔丁基1 - 甲基 - 2 - 丙炔基醚（BMP）对细胞色素CYP2B1 [野生型（WT）]及其苏氨酸205突变为丙氨酸的突变体（T205A）基于机制的失活作用。BPA对WT的失活表现出1343 min⁻¹mM⁻¹的k(inact)/K(I)值和1的分配比。BMP对WT的失活表现出33 min⁻¹mM⁻¹的k(inact)/K(I)值和10的分配比。对WT的液相色谱/串联质谱分析（LC/MS/MS）显示：1）BPA失活导致形成一种蛋白质加合物，其质量增加量相当于BPA加上一个氧原子的质量；2）BMP失活导致形成多个血红素加合物，所有加合物的质量增加量均相当于BMP加上一个氧原子的质量。LC/MS/MS分析表明，谷胱甘肽（GSH）与BMP或BPA的乙炔基部分反应形成GSH缀合物，氧原子添加到内部或末端碳上。对于BPA和BMP对T205A的失活，k(inact)/K(I)值分别被抑制了100倍和4倍，分配比分别增加了9倍和3.5倍。BMP使T205A失活后仅检测到一种主要的血红素加合物。这些结果表明，F - 螺旋中的苏氨酸205在BPA和BMP对CYP2B1基于机制的失活效率中起重要作用。还进行了同源建模和底物对接研究以辅助解释实验结果。
